taxol affects nuclear lamina and pore complex organization ...mediated nick end labeling assays, 4,...

[CANCER RESEARCH 59, 4625–4633, September 15, 1999]

Taxol Affects Nuclear Lamina and Pore Complex Organization and Inhibits Importof Karyophilic Proteins into the Cell Nucleus1

Panayiotis A. Theodoropoulos, Hara Polioudaki, Olga Kostaki, Stavros P. Derdas, Vassilis Georgoulias,Catherine Dargemont, and Spyros D. Georgatos2

Departments of Basic Sciences [P. A. T., H. P., O. K., S. P. D., S. P. G.] and Internal Medicine [V. G.], The University of Crete, School of Medicine, 71 110 Heraklion, Crete,Greece; and Institut Curie, UMR 144, 75248 Paris Cedex 05, France [C. D.]

ABSTRACT

Treatment of human carcinoma cells with Taxol induces focal unrav-eling of the nuclear lamina and extensive clustering or ectopic localizationof the nuclear pore complexes. These striking aberrations develop whenthe cells are transferred to drug-free medium and are allowed to completemitosis. As could be confirmed by terminal deoxynucleotidyl transferase-mediated nick end labeling assays, 4, 6-diamidino-2-phenylindole staining,5-bromo-2-deoxyuridine incorporation, and examination of the nuclearlamins by Western blotting, the malformation of the nuclear envelope isnot a consequence of apoptosis or G1 arrest. In fact, Taxol-treated cellspossessing a defective nuclear envelope remain alive and replication-competent for at least 24 h, undergoing programmed death 72 h afterremoval of the drug. While still in the nonapoptotic state, these cells losethe ability to import karyophilic proteins into the nucleus. Diminishednucleocytoplasmic transport through the nuclear pore complex can bereadily demonstrated by in vitro assays involving digitonin-permeabilizedcells or in vivo monitoring of nuclear factor-kB translocation upon stim-ulation with tumor necrosis factor-a. These observations reveal novelcellular targets of antimicrotubule drugs and may pave the way forimproved schemes of anticancer treatment.

INTRODUCTION

Taxol (Paclitaxel) is a plant alkaloid that is commonly used in thetreatment of human carcinomas (1). It acts by stabilizing the cellularmicrotubules, rendering them rigid and less dynamic (2, 3). Thepharmacological effects of the drug vary, depending on dose andtreatment scheme. When administered at low concentrations (10–100nM), Taxol induces mitotic arrest (4, 5), inhibits protein prenylation(6), and leads, eventually, to apoptosis (5–7). At micromolar doses, italso promotes synthesis and release of TNF-a3 (8, 9), expression ofinterleukin 1 (10) and interleukin 8 (11), and activation of earlyresponse genes (12).

Recent observations show that Taxol activates the Raf-1 kinase (13)and promotes phosphorylation of bcl-2, a “guardian of microtubuleintegrity” (14). Increased bcl-2 phosphorylation may weaken bcl-2/bax complexes, unleashing the latter and triggering apoptosis. Inaddition to that, Taxol and other antimicrotubule drugs are known toaffect the shape of the cell nucleus. After exiting mitosis, cells treatedwith taxanes orVinca alkaloids often develop lobulated nuclei andmultiple micronuclei (15). Interestingly, similar defects are detectedwhen mutants of the nuclear matrix protein NuMA (16) or the Ran/TC4 exchange factor RCC1 (17) are expressed in higher eukaryoticcells.

A major determinant of nuclear form and architecture is the nuclearenvelope (18). This is a highly organized assembly comprising: theouter and inner nuclear membrane; the nuclear lamina meshwork, anintermediate filament system that is associated with the inner surfaceof the nucleus; and the NPCs. The outer nuclear membrane representsan extension of the endoplasmic reticulum, whereas the inner nuclearmembrane constitutes a specialized environment that accommodates aunique set of proteins (LBR, LAP1s, and LAP2s). The nuclear lamina,is composed of A- and B-type lamins. These proteins form a poly-meric lining that supports the inner nuclear membrane and impartselasticity to the nuclear envelope. The NPCs are 125-MDa complexescontaining 50–100 distinct polypeptides (nucleoporins). They providethe sole means for regulated transport between the cytoplasm and thenucleoplasm and are conserved in all eukaryotic cells, from human toyeast. The nuclear envelope disassembles during mitosis and reassem-bles at the end of cell division. A number of factors, including thecdc2 kinase and the microtubules, appear to play a role in this process,but the exact mechanism of reversible disassembly has not yet beenelucidated (for review see Ref. 19).

To understand how antimicrotubule drugs affect the cell nucleus,we have examined thein situ organization of the NPCs and thenuclear lamina in human carcinoma cells treated with low concentra-tions of Taxol. Using functional assays, we have also assessedwhether Taxol-treated cells could carry out nucleocytoplasmic trans-port in a normal fashion before they become apoptotic. Observationspresented below show that Taxol induces striking nuclear envelopeaberrations and compromises macromolecular traffic through theNPC.

MATERIALS AND METHODS

Cells and Treatments. Ishikawa cells were maintained in MEM, whereasHeLa cells were grown in DMEM. The various drugs, diluted in culturemedium, were applied for 20 h at 37°C. After treatment, the cells were rinsedwith fresh medium and incubated for 3–72 h under standard conditions.Viability was determined by trypan blue staining and hemocytometer counting.

Indirect Immunofluorescence Microscopy. Fixation, permeabilizationwith Triton X-100, and antibody staining were performed exactly as describedpreviously (20, 21). The antibodies used included: an anti-a-tubulin mono-clonal (clone DM1A), obtained from Sigma Chemical Co. (St. Louis, MO); theantivimentin polyclonal aV2 (22); the antikeratin monoclonal Lu-5, purchasedfrom Boehringer (Mannheim, GmbH, Germany); the antinucleoporin mono-clonal 414 (23); the antinucleoporin p68 autoimmune serum no. 27 (24); ananti-LAP2B polyclonal (21, 25); the anti-lamin B polyclonal no. 16 (20–22);the anti-lamin A polyclonal no. 163 (21, 26); the anti-all-lamin polyclonal aLI(27); the anti-lamin A/C monoclonal XB-10 (21); a polyspecific serum di-rected to multiple endoplasmic reticulum membrane proteins (28); and ananti-NFkB65 polyclonal purchased from Santa Cruz Biotechnology (SantaCruz, CA). Specimens were examined in a Zeiss epifluorescence and a Leicaconfocal microscope.

Video Microscopy. Monitoring of living cells after release from Taxol wasaccomplished using a computer-operated Leica microscope equipped withappropriate low-illumination optics and a stage chamber for culturing cells.

Detection of Apoptosis.TUNEL assays were performed using anin situcell death detection kit obtained from Boehringer Mannheim. Annexin V/pro-

Received 4/9/99; accepted 7/20/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by the Greek Secretariat of Research and Technology(PENED Grant (to S. D. G.); Greek-German Cooperation Grant (to P. A. T.).

2 To whom requests for reprints should be addressed, at The University of Crete,School of Medicine, Stavrakia, 71 110 Heraklion, Crete, Greece. Phone: 30-81-39.45.39;Fax: 30-81-39.47.59; E-mail:[email protected].

3 The abbreviations used are: TNF-a, tumor necrosis factor-a; NPC, nuclear porecomplex; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling;BrdUrd, 5-bromo-2-deoxyuridine; DAPI, 4, 6-diamidino-2-phenylindole; NLS, nuclearlocalization signal; NFkB, nuclear factorkB.

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Fig. 1. Intracellular organization of human carcinoma cells after Taxol treatment. Cells cultured for 20 h in the presence of 10 nM Taxol were fixed either immediately after treatment(a–d) or after removal of the drug and further culture in Taxol-free medium (e–h). Shown here are representative indirect immunofluorescence images depicting the intracellulardistribution of the microtubules (aande), nucleoporins (bandf), lamins A/C (c), and lamins B1/B2 (d,g, andh). a9–h9, corresponding DAPI profiles.Arrowheads(a9, d9, e9, andg9),ectopically localized chromosomes;arrow (g), chromatin fragment that has assembled an elementary lamina around it;arrows (h), very small lamina deficits present in one of the twodaughter nuclei (for further details on this, see Fig. 4 and text).Scale bars, 4mm.

Table 1 Morphometric analysis and viability of Taxol-treated cells

NTa T/0 T/R3 T/R7 T/R14 T/R24 T/R48 TR/R24 VI/R24 T/R72

Mitotic index and nuclear morphologyb

Interphase cells (%) 95.8 28.5 28.9 48.6 75.0 94.9 95.7NLN (%) 93.8 15.2 13.6 18.6 31.3 54.8 52.6LN (%) 1.3 10.6 11.0 22.7 36.3 26.9 31.0MMN (%) ND 2.7 4.2 7.3 7.4 13.1 12.1Mitotic cells (%) 4.9 71.5 71.1 51.4 25.0 5.1 4.3

Nuclear envelope defectsc

Pore clustersNLN (%) 10 10 10LN 1 MMN (%) 99 95 95

Lamina deficitsNLN (%) 30 39 30LN 1 MMN (%) 90 94 88

Viabilityd

Total cells/plate (3105) 4.4 10 11 11Alive cells/plate (3105) 3.6 7 8 5Dead cells (%) 18 27 32 60

a NT, nontreated cells; T, Taxol-treated cells; TR, taxotere-treated cells; VI, vinorelbine-treated cells; 0, R3, R7, R14, R24, R48, and R72, cells released for 0, 3, 7, 14, 24, 48, and72 h, respectively; NLN, normal-looking nuclei; LN, lobulated nuclei; MMN, multiple micronuclei; ND, not detected.

b The mitotic index and the nuclear morphology of cells treated with Taxol for 20 h were established after the cells were released for various periods of time in drug-free mediumand triply stained with DAPI, antitubulin, and anti-lamin B antibodies. Averages of two independent experiments are provided here, totaling at least 1000 cells for each time point.

c Morphometric analysis of nuclear defects was performed by staining the cells 24 h after release from Taxol, taxotere, and vinorelbine with antinucleoporin and anti-lamin Bantibodies. The numbers represent percentages of nuclei possessing lesions in a total of 1000 cells.

d Viability measurements were performed by culturing cells in 35-mm Petri dishes and counting in duplicate after trypan blue staining. The numbers given represent averages from10 independent samples.

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NUCLEAR ABERRATIONS INDUCED BY ANTIMICROTUBULE DRUGS



pidium iodide assays were performed using a kit purchased from Genzyme/Techne (Cambridge, MA).

Cell Proliferation Assays. Passage of cells through the S phase wasassessed by BrdUrd incorporation, using a kit from Boehringer Mannheim.

Immunochemical Methods. Western blotting and immunoprecipitationfrom total cell lysates were performed as specified previously (21).

RESULTS

Mitotic Arrest and Re-entry into the Cell Cycle. Treatment ofhuman endometrial carcinoma (Ishikawa) cells with 10 nM Taxolresulted in a sustained mitotic arrest. After a 20-h incubation,.70%of the cells possessed condensed chromosomes, disassembled NPCs,and depolymerized nuclear laminae (Table 1 and Fig. 1,b–d andb9–d9). A-type lamins were randomly scattered in the cytoplasm,whereas B-type lamins were relatively concentrated at the spindlepoles (compare Fig. 1,c andd; for relevant information see Ref. 20).The geometry of the mitotic apparatus was minimally perturbed(mostly bipolar spindles), and the majority of the chromosomes werealigned at the cell equator (Fig. 1,a and a9). Consistent with ablockade at the metaphase-anaphase transition, prophase and telo-phase figures could not be detected.

Seven to 24 h after removal of Taxol from the medium, numeroustelophase figures appeared in the fields (Fig. 1,e–h), whereas thepercentage of metaphase cells was gradually reduced (Table 1). Abor-tive cytokineses were not evident, but sometimes a nuclear envelopeformed around ectopic chromatin particles that had not segregatedcorrectly (Fig. 1,e, e9, g, and g9, small arrowsand arrowheads).Trypan blue staining showed that the number of live cells nearlydoubles 24 h after release (Table 1), whereas video monitoring con-firmed that these cells physically divide passing through anaphase andtelophase (Fig. 2,a–d).

In line with the current literature, the cells possessed nuclei withvariable morphologies. More specifically, 24 h after removing thedrug, roughly half of the population contained oval-shaped, normal-looking nuclei, whereas the other half contained lobulated or multiplemicronuclei (Table 1). To assess whether these cells progressed nor-

mally through the cell cycle or were arrested at the G1 restrictionpoint, we incubated the cultures with the base analogue BrdUrd. In theabsence of DNA damage (as shown below in Fig. 5a), incorporationof this compound into the nucleus indicates entry into the S phase andsuccessful crossing of the G1-S straits. Irrespective of nuclear pheno-type, the majority of the cells incorporated BrdUrd within 24 h afterremoval of Taxol (Fig. 2,e–g). DNA replication by BrdUrd-labeledcells was confirmed by fluorescence-activated cell sorting analysis(data not shown). From these data, we conclude that, upon releasefrom mitotic blockade, drug-treated cells reenter the cell cycle, effec-tively complete mitosis, and cross the G1 checkpoint.

Structural Aberrations. To examine whether the nuclear enve-lope of Taxol-treated cells was normally organized, we used specificantibodies recognizing NPC proteins (nucleoporins) or nuclearlamins. Immunostaining with antinucleoporin antibodies 24 h afterrelease indicated that;50% of the cells contained abnormally ar-ranged nuclear pores. Whereas the NPCs of nontreated cells wereclosely spaced, yielding a “rim” fluorescence pattern (Fig. 3a), thoseof the treated cells were organized in large clusters with pore-freeareas in between (Fig. 3,b andc). A more systematic survey of thespecimens by confocal microscopy confirmed the existence of poreclusters at the nuclear periphery (Fig. 3,e–g) and further revealed thatNPC material was sometimes deposited in the cytoplasm (Fig. 3d). Asindicated by morphometric data (Table 1), NPC clustering was morefrequently seen among cells with lobulated nuclei and multiplemininuclei. Interestingly, defects in NPC distribution could be de-tected as early as 1 h after release from Taxol.

Staining with anti-lamin B antibodies revealed numerous “gaps” inthe nuclear lamina meshwork of both normal-looking and irregularlyshaped nuclei. The lamina deficits imparted a “moth-eaten” appear-ance to the nuclear lamina and could be better assessed by inspectingthe specimens at different focal levels (Fig. 4,b–d and b9–d9). Ingeneral, the lamina deficits detected in micronuclei were more exten-sive than in other types of nuclei, yielding “crescent-like” patterns(Fig. 4e). This kind of lesions were never detected in nontreated cellsthat possessed a continuous nuclear lamina meshwork investing pre-

Fig. 2. Fate of human carcinoma cells after release from Taxol.a–d, consecutive video captures showing a cell that undergoes mitosis 5 h after release from Taxol (phase contrastmicrographs).Arrows (a–d), the metaphase plate, chromosome packages, and daughter cell nuclei.e–g, incorporation of BrdUrd in cells treated with Taxol and released for 24 h intonormal medium. BrdUrd has been traced using anti-BrdUrd antibodies and indirect immunofluorescence microscopy.Arrowheads(e), cells that have incorporated various amounts ofthe base analogue into the DNA, having progressed to a different extent through the cell cycle. As can be seen inf andg, lobulated nuclei and micronuclei incorporate BrdUrd similarlyto the oval-shaped nuclei shown ine. Scale bars, 5mm.

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cisely the “folds” of the nuclear surface (Fig. 3,a anda9). Althoughless obvious at a first glance, very small lamina gaps could also bediscerned in cells that were in the process of exiting mitosis (Fig. 1,h andh9, arrows), suggesting that these aberrations develop early after

release from mitotic blockade. Immunostaining with other antinuclearenvelope antibodies yielded essentially the same results, showinglesions in the nuclear lamina, which contained both A- and B-typelamins and aberrant distribution of the integral protein LAP2 (data notshown).

To find out whether the alterations of the nuclear envelope werepeculiar to the specific antimicrotubule agent we have been using, werepeated this analysis comparing the effects of Taxol with those oftaxotere (a different taxane) and vinorelbine (aVinca alkaloid deriv-ative). In pilot experiments, we have determined that the cell cycle-arresting effect of 10 nM Taxol could be reproduced by 1–10 nM

taxotere or vinorelbine, making such a comparison meaningful. Thethree antimicrotubule drugs induced nuclear envelope aberrations toalmost the same extent (Table 1). Lamina deficits were detected in alltypes of cells, irrespective of nuclear morphology, whereas NPCclusters were more commonly seen in cells with multiple micronucleior a single lobulated nucleus.

As has been described above, 24 h after removal of Taxol from themedium, the cells divide and pass through the G1 checkpoint. How-ever, as indicated in Table 1, the total number of cells remainsconstant thereafter, whereas the proportion of dead cells reaches thelevel of 60% 72 h after release. Prompted by these findings, we soughtto determine at which point after release programmed cell deathcommences.

Twenty-four h after removal of Taxol, all cells were TUNEL-negative (Fig. 5a). Their nuclei, deformed or normal-looking, werefree of apoptotic lobules, as could be judged by DAPI staining (Fig.5a9). Use of an annexin V assay and staining with propidium iodide,both of which detect early apoptotic phenomena (29, 30), fully con-firmed these observations (data not shown). The same results wereobtained when we examined cells that had been released for 48 h, theonly difference being that a small proportion of them (;10%) wereTUNEL-positive (Fig. 5,b and b9). Eventually, most of the cellsbecame TUNEL-positive 72 h postrelease (Fig. 5c), as the chromatincondensed into lobules and nuclear architecture was abolished (Fig.5c9).

To assess when apoptotic degradation of nuclear envelope proteinsis initiated, we continued with immunoprecipitation and Westernblotting experiments. Cells treated with Taxol and released for 24 h,as well as controls, were extracted with 2% Triton-300 mM NaCl, andthe solubilized proteins were immunoprecipitated with anti-lamin Bantibodies. As shown in Fig. 6a, comparable amounts of B-typelamins could be detected in precipitates from treated and untreatedcells (compareLanesN and24). Western blotting analysis of total cellextracts (Fig. 6c) and isolated nuclei (Fig. 6b) derived from cells thathad been released for 24 h did not reveal any change in the lamin Bpattern, with respect to controls (compareLanesN and24). The samewas observed with cells released for 48 h (Fig. 6d, compareLanesNand48). However, extensive lamin degradation was noted 72 h afterremoval of Taxol (Fig. 6d, compareLanes N and 72), indicatingactivation of ICE proteases. From these and previous morphologicalresults, we can safely conclude that the nuclear lamina and NPCabnormalities detected by immunofluorescence microscopy developwell before the advent of apoptosis.

Functional Defects.Having established that antimicrotubuleagents dramatically alter nuclear envelope organization, we then ex-amined whether Taxol treatment affects macromolecular trafficthrough the NPC. To assess nuclear import in a controlled fashion, weused a well-established,in vitro system (31). Intact cells, eithernontreated or treated with Taxol and released for 24 h into normalmedium, were permeabilized by low concentrations of digitonin, anagent known to open the plasma membrane without affecting theintegrity of the nuclear envelope. After washing out all cytosolic

Fig. 3. Aberrant distribution of the NPCs.a anda9, nontreated cells.b–g,b9, andc9,cells treated with Taxol and cultured for 24 h into normal medium. The specimens havebeen stained with antinucleoporin antibodies (a–g) and DAPI (a9–c9). a–c and a9–c9,conventional epifluorescence pictures;d–g, confocal images.Arrows(b andc), large NPCclusters which contrast the continuous, rim-fluorescence pattern seen ina. The micrographin d shows ectopically localized NPCs that have accumulated in the cytoplasm.e–g,consecutive optical sections of the same cell, illustrating the distribution of NPC clusterson the surface of the nucleus.Scale bar, 5mm.

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constituents, the resulting “ghosts” were supplemented with aXeno-pus laevisegg extract (which contains soluble import factors), energy(ATP, GTP, and ATP-regeneration system), and FITC-labeled BSAcoupled to the SV40 large T antigen NLS. As shown in Fig. 7b, ghostsderived from nontreated cells efficiently imported the karyophilicligand into the nucleus. However, ghosts prepared from Taxol-treatedcells did not accumulate BSA-NLS into the nucleus under the sameassay conditions (Fig. 7c). Because, in both cases, the necessarysoluble factors were provided exogenously, this result strongly sug-gests that it is NPC itself and not the cytosolic transport machinerythat is primarily affected by Taxol treatment. Nuclear import under theconditions of the assay was specific and did not occur whenXenopuscytosol was omitted from the reaction (Fig. 7a). The inability of

Taxol-treated cell models to accumulate BSA-NLS into the nucleuswas not due to nuclear envelope lysis during preparation of the ghosts.This could be confirmed by permeabilizing Taxol-treated cells withdigitonin and probing with antibodies that recognize nucleoplasmi-cally disposed antigens (e.g., lamins). In this setting, nuclear laminastaining was not observed, indicating that the nuclear membraneswere intact (data not shown; for relevant data see Ref. 20).

On the basis of thesein vitro findings, we further asked whetherTaxol affects nucleocytoplasmic transport underin vivo conditions.To monitor import of macromolecules in living cells, we studied thepartitioning of the transcription factor NFkB, which is rapidly trans-located from the cytoplasm to the nucleus when cells are exposed toTNF-a. Taxol-treated cells and nontreated controls were first incu-

Fig. 4. Nuclear lamina defects.a, a9, and a0,nontreated cells.b–e,b9–e9, andb0-d0, cells treatedwith Taxol and cultured for 24 h into normal me-dium. The specimens were stained with anti-laminB antibodies (a–eanda9–d9) and DAPI (a0–d0 ande9). Conventional epifluorescence pictures taken atdifferent focal levels are shown ina–danda9–d9 tobetter visualize nuclear lamina damage. Note theinterruptions of the normal rim-fluorescence pat-tern and the moth-eaten appearance of the structure(arrowheadsandarrows).Scale bar, 3mm.

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bated in serum-free medium and then stimulated with 10 ng/ml humanTNF-a. After 30 min, they were fixed, and the location of NFkB wasdetermined by indirect immunofluorescence microscopy. Neither nor-mal (Fig. 8, a and a9) nor Taxol-treated (Fig. 8,c and c9) cellsimported NFkB in the absence of TNF-a stimulation. However, afterexposure to TNF-a, all of the nontreated cells imported NFkB into thenucleus (Fig. 8,b and b9). This did not happen with Taxol-treatedcells. In that case, whereas the majority (;90%) of the cells with anormal-looking nucleus successfully translocated NFkB across thenuclear envelope, virtually all of the cells that contained multiplemicronuclei and lobulated nuclei failed to do so (Fig. 8,d andd9). Thisresult seems to agree with the fact that NPC defects are much morefrequently seen among cells with aberrantly shaped or sized nucleithan among cells with a single, normal-looking nucleus (Table 1).

DISCUSSION

Mechanisms of Taxol-induced Defects.We have shown here thatTaxol and other antimicrotubule agents affect nuclear envelope orga-nization and compromise nucleocytoplasmic transport. Clustering ofthe NPCs associated with nuclear lamina aberrations has also beenseen in neuronal cells ofDrosophila melanogasterupon disruption oftheDm0 lamin gene (32), whereas pore clustering and mRNA exportdefects have been detected in yeast strains expressing mutant nucleo-porins (Ref. 33 and references therein). Whether these defects and theaberrations caused by antimicrotubule agents share a common patho-physiological basis is not clear at this time.

Apoptotic cleavage of the nuclear lamins upon treatment of humanbreast carcinoma cells with Taxol has been reported by McCloskeyetal. (34). However, our observations in human endometrial and cervi-cal carcinoma cells indicate a different type of defect that developswell before degradation of nuclear envelope constituents. The possi-bility that the Taxol-induced defects arise from apoptosis can be ruledout because TUNEL and annexin V assays do not reveal early alter-ations in DNA structure or plasma membrane organization at timepoints when the NPCs and the nuclear lamina are clearly disorganized.

Furthermore, physical degradation of nuclear lamins, which is diag-nostic for ICE protease activation, is not detected until 72 h afterrelease. Although we cannot exclude by the currently available tech-niques that a small amount of the lamins (i.e., 1–2%) are proteolyzed

Fig. 6. Apoptotic degradation of B-type lamins.a, detergent/high-salt extracts ofcontrol (Lane N) and Taxol-treated cells released for 24 h (Lane 24) were precipitated byanti-lamin B antibodies. The immunoprecipitates have been resolved by SDS-PAGE andprobed with the same antibodies.b–d, Western blotting analysis of nuclear extracts (b)and total cell lysates (c andd) from control (Lanes N) and Taxol-treated cells released for24 (Lanes 24), 48 (Lane 48), and 72 (Lane 72) h. The profiles shown represent threeseparate Western blotting experiments. Blots ina and b were developed by alkalinephosphatase-conjugated secondary antibodies, whereas those inc and d have beendeveloped by ECL. Both B1 (Mr 68,000) and B2 (Mr 74,000) lamins are detected by themore sensitive chemiluminescence technique, whereas only the major lamin B1 band isvisible by color.Arrows, position of unproteolysed lamins;bracket, major degradationproducts generated by apoptotic cleavage;arrowheads, minor degradation fragments thatarise fromin vitro proteolysis during sample processing;p, heavy chain of IgG.Tx-IP,immunoprecipitation from Triton X-100/high-salt extract;Ncl-Blot, Western blot onisolated nuclei;Tot.Ex.Blot, Western blot on total cellular extract.

Fig. 5. Apoptotic manifestations after release from mitotic blockade.a–c, TUNEL profiles 24, 48, and 72 h, respectively, after release from Taxol;a9–c9, corresponding DAPIprofiles. Note the TUNEL-positive staining 72 h after release.Scale bar, 4mm.

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early after treatment, it is important to note that most the treated cellsdivide successfully, pass the G1 checkpoint, and transit through the Sphase after release into normal medium. This would be unlikely forcells that have initiated the process of programmed death.

Considering the current literature, one could speculate that nuclearenvelope lesions develop as a result of decreased farnesylation of thenuclear lamins. Although this would be consistent with the inhibitoryeffects of Taxol on protein prenylation (6), it does not fit the fact thatlamins assemble normally when isoprenylation is blocked by thespecific farnesyl transferase inhibitor BZA-5B (35).

Nuclear lamina and NPC alterations could also be a consequence ofincreased phosphorylation mediated by mitogen-activated protein ki-nase and other protein kinases that are activated by antimicrotubuledrugs (36). Along the same lines, the structural abnormalities inducedby Taxol are morphologically similar to microtubule-dependent lam-ina lesions and nuclear shape irregularities that develop in fibroblasticcells overexpressing a dominant-negative form of the ATR kinase(37) after treatment with hydroxyurea. ATR, a protein related to theataxia-telengiectasia gene product (ATM), is a member of the phos-phatidylinositol kinase family and an important component of severalcell cycle checkpoints (38). Whether nuclear envelope proteins con-stitute an end target of these enzymes is an exciting possibility thatneeds to be addressed in future studies.

Apoptosis and metabolic effects aside, we should finally considerthe consequences of altered microtubule dynamics on nuclear enve-lope reassembly because taxanes andVinca alkaloids, when given at

nanomolar doses, lower dynamic instability without significantly af-fecting the mass of tubulin polymer inside eukaryotic cells (15). Whenadministered at a dose of 10 nM, Taxol is taken up and concentrated.800-fold by binding to cellular microtubules (4, 5). At this concen-tration (;8 mM), the drug decreases exchange of tubulin subunits atpolymer ends (dynamicity) and blocks cells in mitosis. Upon releaseinto normal medium, the intracellular concentration of Taxol is grad-ually reduced, allowing reentry into the cell cycle. However, whereasthe intracellular concentration of Taxol is still rather high (;4 mM

according to Ref. 5), it is clear that most cells begin to divide.Knowing this, we are bound to think that nuclear envelope aberrationsmay develop because partially stabilized microtubules, contrary totheir normal counterparts, fail to “unload” important components of

Fig. 7. In vitro import assay.a anda9, nontreated cells permeabilized with digitoninand incubated with BSA-NLS in the absence ofXenopuscytosol; b and b9, nontreatedcells permeabilized with digitonin and incubated with BSA-NLS in the presence ofXenopuscytosol; andc andc9, Taxol-treated cells 24 h after release permeabilized withdigitonin and incubated with BSA-NLS in the presence ofXenopuscytosol. For details,see text.a–c, FITC fluorescence;a9–c9, DAPI staining.Scale bar, 10mm.

Fig. 8. Nuclear translocation of NFkB. a anda9, nontreated cells before stimulationwith TNF-a; b and b9, nontreated cells after stimulation with TNF-a; c and c9, Taxol-treated cells released for 24 h before stimulation with TNF-a; andd andd9, Taxol-treatedcells released for 24 h after stimulation with TNF-a. For details, see text.a–d, location ofNFkB65 subunit;a9–d9, DAPI staining. All images were obtained using a confocalmicroscope.Arrowheads, cells that have failed to import NFkB in a normal fashion.Scalebar, 10 mm.

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the nuclear envelope and the nuclear matrix that transiently dock onthe mitotic spindle during metaphase and anaphase (16, 20, 21, 39,40). The timely release of these factors from the microtubules isprobably necessary to assemble anew the nucleus of the daughter cellsin the end of the division cycle.

Impairment of Nuclear Import and Induction of Apoptosis.The observations presented here substantiate the idea that microtu-bule-acting agents impair nuclear import. Looking critically at ourimport data, we should note that nuclear transport in Taxol-treatedcells is universally inhibited underin vitro conditions because nuclearaccumulation of the karyophilic substrate (BSA-NLS) is abolished inall types of digitonin ghosts. However, in anin vivo setting, cells withmicronuclei and lobulated nuclei are primarily affected, whereas thosethat possess normal-looking nuclei are influenced much less from afunctional point of view. From this, we suspect that NPC abnormal-ities develop in all cells but are perhaps more extensive and moreeasily identifiable in those with an aberrant nuclear morphology. Itcould also be that NPC malfunction is much more pronouncedin vitrobecause auxiliary or repairing factors have been removed, and thesystem is challenged to perform under significantly more demandingconditions.

Finally, at the conclusion of this communication, we ought todiscuss the effects of Taxol on NFkB function. Recent studies haveestablished that NFkB is a major antiapoptotic factor in mammaliancells. Liver cell apoptosis followed by embryonic lethality is observedin RelA (NFkB65)-deficient mice, whereas fibroblasts from RelA-null animals are extremely sensitive to apoptotic stimuli such asTNF-a treatment, ionizing radiation, and treatment with daunorubicin(41, 42). NFkB induces expression of theTRAF1,TRAF2, c-IAP1,and c-IAP2genes, which produce potent suppressors of caspase-8(43). Knowing these observations, we would like to propose thatinhibition of NFkB translocation by antimicrotubule drugs removes amajor line of antiapoptotic defense and renders the cells prone toprogrammed death. This postulate is fully supported by recent studieson mice showing an additive or supra-additive effect of Taxol andTNF-a treatment (44). From the sum of these data, it can be predictedthat combined therapy schemes that exploit the proapototic propertiesof TNF-a (45), the NFkB-inactivating potential of corticosteroids orsalicylates (46–48), and the import-inhibiting ability of taxanes (thisreport) might enhance the efficacy of anticancer treatment.

ACKNOWLEDGMENTS

We thank A. Demakopoulos (The University of Crete, Heraklion, Greece)for help in experimental work and C. Maison, J-B. Sibarita, and J. Salamero(Institut Curie, Paris, France) for advice and materials. We are also indebted toR. Hartig and P. Traub (Max Planck Institute, Ladenburg, Germany) forallowing the use of their confocal microscope.

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1999;59:4625-4633. Cancer Res Panayiotis A. Theodoropoulos, Hara Polioudaki, Olga Kostaki, et al. Nucleusand Inhibits Import of Karyophilic Proteins into the Cell Taxol Affects Nuclear Lamina and Pore Complex Organization

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