[CANCER RESEARCH 61, 1220–1226, February 1, 2001]

Intranuclear Compartmentalization of Cyclin E during the Cell Cycle: Disruptionof the Nucleoplasm-Nucleolar Shuttling of Cyclin E in Bladder Cancer1

Gloria Juan and Carlos Cordon-Cardo2

Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT

Cyclin E/cyclin-dependent kinase 2 complexes are essential during thecell cycle for entrance into S phase. Cyclin E expression starts in mid-G1,reaches a maximum at S-phase entrance, and undergoes proteolysis me-diated by the ubiquitin pathway as the cell progresses through S phase.Laser scanning cytometry, a microscope-based cytofluorometer combin-ing the advantages of both flow and image analysis, allowed the determi-nation of subcellular localization of cyclin E, p27, and retinoblastomaprotein during cell cycle progression in normal human fibroblasts andnine bladder cancer cell lines. We observed that in normal fibroblasts andmost tumor cell lines, cyclin E localizes in the nucleoplasm during mid-G1and is translocated to the nucleolus during G1-S-phase transition, and itslevels are undetectable in G2-M phase. Neither levels nor subcellularlocalization of p27 and retinoblastoma protein was cell cycle dependent innormal or tumor cells. However, four of nine bladder cancer cell linescontinued to express cyclin E in all phases of the cycle, and image analysisrevealed that it was localized to nucleoli. These observations suggest thatthe nucleolus mediates a cyclin E “shuttling” between the nucleus and thecytoplasm that is probably involved in its regulation and that this mech-anism could be disrupted in bladder cancer.

INTRODUCTION

Cell cycle progression is controlled by protein complexes com-posed of cyclins and Cdks3 (1–6). These complexes phosphorylatefundamental elements involved in cell cycle transitions, such as thepRB. Phosphorylation of pRB releases E2F members, which activatetranscription of the components of the DNA replication machinery,thereby committing the cell to S phase (7, 8). The expression ofdistinct cyclins occurs at specific and well-defined periods of thecycle. Cyclins D1–D3 and E are maximally expressed during G1

phase, regulating the transition from G1 to S phase. Cyclins A, B1,and B2 reach their maximal levels during S and G2 phases and areregarded as regulators of the transition to mitosis. Cyclin D1/Cdk4complexes govern G1 progression, whereas cyclin E/Cdk2 complexescontrol entry into S phase. Cyclin A/Cdk2 complexes regulate passagethrough S phase, and cyclin B/Cdc2 control entry into mitosis (1).Cyclins are regulated not only at the transcriptional and translationallevels but also by their rate of degradation via the ubiquitin pathway(9, 10). An additional level of negative control is produced by theexpression of Cdk inhibitors. Cdk inhibitors have been classified intotwo groups: (a) KIPs (kinase inhibitory proteins; p21CIP1, p27KIP1,and p57KIP2); and (b) INKs (inhibitors of kinases; p15INK4b, p16INK4a,p18INK4c, and p19INK4d; Refs. 1–8).

Immunocytochemical detection of cyclins in combination withanalysis of DNA content by multiparameter flow cytometry allows

correlation between the presence of a particular cyclin and the posi-tion of the cell in the cycle (11–15). Expression studies performed inasynchronous cell populations do not perturb cell cycle progressionand do not induce growth imbalance, both of which otherwise occurwhen cells are synchronized (16, 17). Moreover, analyses of asyn-chronous populations can reveal intercellular variability in cyclinexpression, detect rare events and cell subpopulations with distinctfeatures, and may determine the presence of thresholds in expressionof these proteins at particular phases of the cell cycle (11, 18).

It has been reported that expression of critical cell cycle regulatorsis frequently altered in human cancer (1–3). To investigate the rela-tionship between subcellular localization of cyclin E, p27, and pRBand cell cycle progression, we have used LSC to analyze normalhuman fibroblasts and nine bladder cancer cell lines. Similar to flowcytometry, LSC allows bivariate analysis of immunocytochemicallylabeled cells (19, 20). In addition, LSC allows the morphologicalidentification of cells whose fluorescence has been measured and theintracellular distribution of such a fluorescence signal.

MATERIALS AND METHODS

Cell Lines. Human bladder cancer cell lines (RT4, T24, J82, HT1197,HT1376, 5637, UMUC3, TCCSUP, and SCABER) and human fibroblasts(CRL-1502) were obtained from American Type Culture Collection (Manas-sas, VA). The cells were cultured as recommended by American Type CultureCollection. All media, supplements, and antibiotics were obtained from theMedia Laboratory Core Service at Memorial Sloan-Kettering Cancer Center.The cultured cells were tested periodically forMycoplasmainfection. Tomaintain asynchronous exponential growth, cells were passaged by dilutingthem to a concentration of 13 105 cells/ml and were repassaged beforeapproaching a density of 53 105 cells/ml. For the experiments, cell mono-layers were grown directly on microscope chamber slides.

Antibodies. The following antibodies were used for the present study. Apurified mouse antinucleolin mAb, clone 4E2, was obtained from ResearchDiagnostics Inc. (Flander, NJ). Two different anti-cyclin E mAbs were used:(a) purified cyclin E mAb clone HE-12 (PharMingen, San Diego, CA); and (b)purified cyclin E mAb clone AB-1 (Oncogene Research). A purified anti-p27mAb, clone G173-524 (PharMingen), was also used. To assess pRB expressionand phosphorylation status, we used two antibodies: (a) anti-pRB mAb clone3C8 (PharMingen), which detected underphosphorylated and hyperphospho-rylated products; and (b) anti-pRB mAb clone G99-549 (PharMingen), whichdetected underphosphorylated pRB.

Immunocytochemical Detection of Nucleolin, Cyclin E, p27, and pRB.The cells grown as monolayers on microscope chamber slides were washed inPBS and fixed in ice-cold 80% ethanol for up to 24 h. After fixation, the slideswere washed twice with PBS containing 1% BSA and 0.1% sodium azide,followed by permeabilization with 0.25% Triton X-100 in PBS on ice for 5min. After that, the slides were incubated with 100ml of PBS containing 0.5mg of the mAb and 1% BSA for 2 h at room temperature. Mouse anti-IgGserved as isotype negative control. The slides were then rinsed with PBScontaining 1% BSA and incubated with 100ml of FITC-conjugated goatantimouse F(ab9)2 (DAKO, Carpinteria, CA) diluted 1:30 in PBS containing1% BSA for 30 min at room temperature in the dark. The slides were washedagain, resuspended in 5mg/ml PI (Molecular Probes) and 0.1% RNase A(Sigma) in PBS to counterstain cellular DNA, and incubated at room temper-ature for 20 min before measurement. The slides were then mounted undercoverslips and analyzed by LSC. Additional details of the staining protocolsare presented elsewhere (12, 20–25).

Received 6/29/00; accepted 11/20/00.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 Supported in part by National Cancer Institute Grant CA-47538.2 To whom requests for reprints should be addressed, at Division of Molecular

Pathology, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275York Avenue, NY 10021. Phone: (212) 639-7746; Fax: (212) 794-3186; E-mail: cordon-c@mskcc.org.

3 The abbreviations used are: Cdk, cyclin-dependent kinase; LSC, laser scanningcytometry; pRB, retinoblastoma protein; mAb, monoclonal antibody; PI, propidiumiodide.

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LSC Setting and Fluorescence Measurement.Fluorescence of individualcells was measured by LSC (CompuCyte, Cambridge, MA). The cellularfluorescence of PI and FITC was excited at 488 nm, and emission of thesefluorochromes was measured using the standard long-pass (570 nm) or band-pass (530 nm) filters, respectively. At least 3000 cells were measured per slide.The relocation feature of LSC (26) was used to identify individual cells withinthe gated populations and to reveal their morphology. Each experiment wasrepeated at least three times. The samples were photographed using the LSCcharge-coupled device camera and/or a Kodak DC120 Zoom Digital Camera(Eastman Kodak, Rochester, NY) adapted to the LSC microscope.

RESULTS

Detection and Quantification of Nucleoli by LSC. To detect andanalyze nucleoli, we developed a novel assay using a cytometricprogram for fluorescencein situ hybridization. Nucleoli were identi-fied by the immunoreactivity of antinucleolin antibodies and visual-ized by the signal of the secondary antibodies conjugated to FITC.

Using this approach, we were able to count the number of “spots”corresponding to nucleoli present inside the nucleus of each individualcell (Fig. 1A).

Fig. 1 illustrates the strategy used to measure the nucleolin-associ-ated FITC fluorescence separately in the nucleoli and the cell nucleus

by LSC. The triggering threshold (“threshold contour” inred) was seton red fluorescence of cells in which the DNA was counterstainedwith PI. The green fluorescence (integrated value) was then measuredin two distinct areas. The first area, corresponding to the nuclear mass,was within the triggering threshold plus four pixels toward the outsideof the threshold (“integration contour” ingreen). This area coveredthe whole cell nucleus. The second area, corresponding to “fluores-cencein situhybridization spots” and shown inwhite in Fig. 1, allowsus to count the number of spots inside the nucleus and corresponds tonucleolar bodies. It also allows the integration of the green fluores-cence signal over the nucleoli. Background fluorescence was auto-matically measured outside the cell (shown inblue) and subtracted.

Subcellular Localization of Cyclin E during the Cell Cycle inBladder Cancer. To study the subcellular localization of cyclin Eduring cell cycle progression, we compared its pattern of expressionin normal fibroblasts and nine bladder cancer cell lines (Fig. 2).

Initial fluorescence microscopic examination of asynchronous, ex-ponentially growing fibroblasts and most cell lines revealed that themajority of cells did not express cyclin E (only ared nuclear signalcorresponding to the PI counterstaining of nuclei; see Fig. 2A). How-ever, a subpopulation of cells showed anorangenucleoplasm pro-duced by the colocalization of cyclin E (green) and PI (red; Fig. 2A).

Fig. 1. Immunocytochemical detection ofnucleolin and quantification of nucleoli by LSC.A,J82 cells were indirectly stained with antinucleolinmAb conjugated to FITC (green), and their DNAwas counterstained with PI (red), as described in“Materials and Methods.” The triggering thresholdis depicted inred, whereas the total nuclear fluo-rescence contour is set ongreen. The nucleolarbodies are contoured inwhite inside the nucleus.See “Results” for more details. Original magnifi-cation,3400.B, the LSC software allows countingof the number of nucleolar bodies (“spots”) presentinside the nucleus of each individual cell. Thebargraph on the left illustrates the number of spots(nucleoli) per cell analyzed. The numerical assess-ments on theright sideof the figure summarize theactual counting of nucleoli (green number of spots).The cells were categorized based on the number ofspots identified per cell and given a region number(i.e., region 1 included 8.3% of cells displayingonly one spot; region 5 included 3.3% of cellsdisplaying 5 spots).Rgn. #, the number of theregion in the graph;Count, the number of events inthe corresponding region;Pct., the percentage of allevents in the region;Mean, the mean value of theevents in the region;FWHM, the coefficient ofvariation calculated based on full width of the dis-tribution of events for a feature at half maximumvalue of the count.

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Fig. 2. Immunocytochemical detection of cyclin E.A, J82 cells stained with anti-cyclin E mAb (FITC) and PI (red), as described in “Materials and Methods.” Note the heterogeneityof the pattern of cyclin E immunostaining in the field, including negative cells (nuclei stainedredonly, due to PI staining),yellow-greennucleoplasm-stained cells (cyclin E colocalizedin the nuclei with PI), and some cells displaying nucleolargreenfluorescence signal (cyclin E expression in nucleolus). Original magnification,3400.B, cytometric analyses of cyclinE versusDNA content on J82 cells. This process allows the dynamic visualization of cyclin E expression in relation to cell cycle position. In addition, gating cells for each cyclecompartment allows us to ascribe subcellular cyclin E localization. In the J82 cells, cyclin E expression is found in the nucleoplasm during mid-G1 phase of the cell cycle and is present

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More interestingly, some cells showed an intenseyellow-greenstain-ing in their nucleoli, representing cyclin E accumulation in thiscellular compartment (Fig. 2A). Similar results were obtained by theuse of the two anti-cyclin E mAbs to distinct epitopes.

We then generated cytometric data analyzing the same cells andrelating their cyclin E profile with cell cycle progression (Fig. 2B).Fibroblasts and certain bladder cancer cell lines, including J82, 5637,T24, and RT4, presented the expected pattern of cyclin E expression.Maximal levels of cyclin E were in cells undergoing transition fromG1 to S phase, and this expression level continuously decreased duringprogression through S phase and was undetectable during G2-Mphase. In addition, a threshold of cyclin E expression was apparent atthe G1-S-phase transition. This approach also allowed us to determinethe relationship between cell cycle and intranuclear compartmental-ization. To achieve this goal, several gates were created aroundpopulations in G0-early G1, mid-G1, G1-S-phase transition, and G2-Mphase, and, using the relocation feature of the LSC, we identifiedindividual cells within the gated populations. The cells in G0-early G1

were cyclin E negative, whereas mid-G1 cells expressed cyclin E inthe nucleoplasm. However, cells in the G1-S-phase transition dis-played a strong nucleolar cyclin E expression, with decreased nucle-oplasmic signals. Finally, G2-M-phase cells had undetectable cyclin Elevels. Fig. 2Billustrates such expression patterns and subcellularcompartmentalization in J82 cells.

Unexpectedly, the analysis of SCABER cells, a cell line derivedfrom a squamous cell carcinoma of the bladder, showed a differentexpression profile (Fig. 2C). We observed that cyclin E in these cellswas present in the nucleoli at all times, regardless of the cell cyclephase, with minimal nucleoplasmic levels. Moreover, the bivariatedistribution of cyclin E in relation to cell cycle position was disturbed,and cyclin E expression did not decrease during S phase, continued inG2-M phase (Fig. 2C), and was mainly nucleolar. Using the cytomet-ric approach and color gating, we further validated this abnormalcyclin E expression profile (Fig. 2D). A gate was set around thecounted nucleoli. As is evident in the histogram of J82 cells, thelabeled population within this gate mostly represented cells in the

Fig. 3. Immunocytochemical detection of p27.Cells were indirectly stained with anti-p27 mAbconjugated to FITC (green), and their DNA wascounterstained with PI (red), as described in “Ma-terials and Methods.”A, CRL-1502 normal humanfibroblasts displaying ayellow-greennuclear fluo-rescence, indicating that p27 colocalizes with PI inthe nuclei. Note a discretegreenpunctate patternthroughout the nucleous.B, 5637 bladder cancercells showing strong nuclear and cytoplasmic im-munostaining.C, J82 bladder cancer cells werefound to have undetectable levels of p27.D,UMUC3 bladder cancer cells were found to have asimilar pattern of p27 expression as 5637 cells.Original magnifications,3400.

in the nucleoli during G1-S-phase transition (for more details, see “Results”). These analyses take advantage of the cytometric quantification and the relocation features of the LSCsoftware.C, cytometric analyses of cyclin EversusDNA content on SCABER cells. These studies were conducted as described above. Note that in these cells, cyclin E is expressedat all times in the nucleoli, following an “unscheduled” pattern of expression, and is not degraded during late S and G2-M phases.D, cytometric detection of cyclin E expression inthe nucleolus during the cell cycle (left panel, J82 cells;right panel, SCABER cells). This is achieved by gating the counted spots, which correspond to nucleolar bodies, andrepresenting such events ingreenon the scattergrams. Note that whereas theleft scattergramand cell cycleinset(corresponding to J82 cells) show cyclin E nucleolar expression onlyduring G1-S-phase transition, theright scattergramand cell cycleinset(SCABER cells) show cyclin E nucleolar expression throughout the cell cycle. Original magnifications,3400.

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Fig. 4. Immunocytochemical detection of pRB.A, normal human skin fibroblasts, CRL-1502, were stained using mAb G99-549 to detect underphosphorylated pRB. Note that thereare two main phenotypes: those cells that react with this G99-549 (greenfluorescent nuclei, representing underphosphorylated pRB); and those with undetectable staining (redstainingdue to PI, mainly expressing hyperphosphorylated pRB products). (Note: this photograph was taken using the LSC charge-coupled device camera rather than with the Kodak DC120Zoom Digital Camera used for the other figures). The bivariate distribution of underphosphorylated pRBversusDNA content shows that most of the reactive cells are in G1 phase.B, normal human skin fibroblasts, CRL-1502, were also stained using mAb 3C8 to detect all pRB products, including hypophosphorylated and hyperphosphorylated pRB proteins. Notethat all cells reacted with this antibody. The bivariate distribution of total pRBversusDNA content shows that pRB is expressed throughout the cell cycle. Original magnification,3400.

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G1-S-phase transition, whereas in SCABER cells, cyclin E levels wereobserved throughout the cell cycle (Fig. 2D). These alterations incyclin E expression and subcellular distribution were also observed inHT1197, TCCSUP, and UMUC3 cells.

Expression Patterns of p27 during Cell Cycle Progression.Incultured fibroblasts, we found high p27 levels in the nuclei of all cells,regardless of their cell cycle position (Fig. 3,A, B, andD). p27 wasfound to have a punctate pattern of nuclear expression in fibroblasts(Fig. 3A). However, p27 was not detected in some of the bladder celllines analyzed. Only J82 cells displayed low to undetectable p27nuclear levels, without cytoplasmic staining (Fig. 3C). p27 immuno-reactivity in the nucleolus was not identified in either normal fibro-blasts or tumor cell lines.

Patterns of Expression and Phosphorylation Status of pRBduring Cell Cycle Progression.Expression levels of pRB do notchange during the cell cycle; however, its activity is regulated byphosphorylation. pRB is hypophosphorylated in quiescent and earlyG1 cells and hyperphosphorylated throughout the rest of the cell cycle.In cultured normal fibroblasts, we observed that underphosphorylatedpRB, recognized by mAb G99-549, is present in all G0-G1 cells witha diffuse nucleoplasmic pattern. Cells that progress through S phaseand G2-M phase revealed low to undetectable levels of underphos-phorylated pRB (Fig. 4A). This pattern is in contrast to that producedby identification of both underphosphorylated and hyperphosphory-lated pRB by mAb 3C8. In bivariate analysis of hyperphosphorylatedpRB versuscellular DNA content, pRB products are present through-out the cell cycle, being more prominently expressed in G2-M-phasecells (Fig. 4B). Bladder cancer cell lines displayed a very differentpattern of pRB reactivity. G99-549 staining was undetectable in allcell lines, but intense nuclear signals were seen for 3C8 mAb, indi-cating that hyperphosphorylated pRB is the prevalent expressedproduct.

DISCUSSION

Cell cycle progression is regulated by a group of heterodimericproteins composed of a cyclin acting as a regulatory subunit and aCdk, which acts as the catalytic subunit. The critical transition fromG1 to S phase is controlled by cyclin E/Cdk2 complexes. Data fromthe present study reveal that normal fibroblasts and most tumor celllines analyzed had undetectable cyclin E levels during G0 and earlyG1 and expressed cyclin E with a nucleoplasmic pattern duringmid-G1, whereas during the G1-S-phase transition, cyclin E localizedto the nucleoli. Cyclin E was absent during G2-M phase (Figs. 2 and5). Interestingly, neither levels nor subcellular localization of p27 was

cell cycle dependent. Similarly, pRB levels and subcellular localiza-tion were also unchanged; however, the phosphorylation status ofpRB was cell cycle dependent.

The shuttling of cyclin E between the nucleolus and the cytoplasmmay serve as a titration mechanism, presenting cyclin E for enzymaticdegradation and allowing progression through the cell cycle. Support-ing this hypothesis, nucleoplasmic-nucleolar shuttling has been re-cently described as a regulatory p53 mechanism involving mdm2-p19Arf in murine models. It has been reported that nucleolar-cytoplasmic shuttling of mdm2 is essential for the ability of mdm2 topromote p53 degradation. p19Arf is encoded at the Ink4a locus andacts by attenuating mdm2-mediated degradation of p53 (27–29).mdm2 protein colocalizes with p19Arf in the nucleolus, and it has beenpostulated that p19 blocks the nucleolar-cytoplasmic shuttling ofmdm2, thus stabilizing p53 (30, 31).

Unexpectedly, four of the nine bladder cancer cell lines (SCABER,HT1197, TCCSUP, and UMUC3) showed an altered pattern of sub-cellular localization of cyclin E. In these cells, cyclin E levels wereconstantly elevated, regardless of the cell cycle position, although weobserved that these cells had a short G1 phase. In addition, cyclin Eexpression was identified in the nucleoli of all cells analyzed. Thesedata suggest that cyclin E in these cells is not exported to thecytoplasm, where it undergoes enzymatic degradation. Because pRBis hyperphosphorylated in these cells at all times, it is tempting topostulate that this deregulated cyclin E profile serves as an oncogenicevent.

Cyclin E has been reported to be overexpressed and to have anunscheduled pattern of expression in certain human tumors, includingbladder cancer (11, 32–37). In addition, the shorter G1 phase observedin certain bladder cancer cells has also been described for humanpapillomavirus type 16-infected cells (38). In those cells, cyclin Eexpression was maintained at high levels in S phase and G2-M phase(38). Furthermore, the cyclin E promoter has an E2F1 binding motif,which appears to be the main transcription control mechanism of itsexpression (39). Because hyperphosphorylated pRB releases E2F1products, this phenomenon could also contribute to the maintenanceof high cyclin E levels. It is not understood how alterations in theexpression of cyclin E contribute to tumorigenesis. A recent study bySpruck et al. (40) reported that down-regulation of cyclin E/Cdk2kinase activity after the G1-S-phase transition may be necessary forthe maintenance of karyotypic stability. Altered shuttling of cyclin Emight also be involved in such a deregulated process in humantumors.

The nucleolus has distinctive involvements such as rDNA localiza-tion and rRNA synthesis. However, it appears that many other func-tional and regulatory events take place in this organelle, includingnuclear export of rRNAs and ribosomal proteins (41–43). Morerecently, a more active role has been attributed to the nucleolus in thetemporarily ordered expression and proteolytic events that occur dur-ing cell growth (44–47). Based on data from this study, we postulatethat the nucleolus mediates a “shuttling” between the nucleus and thecytoplasm to control cyclin E levels and that this mechanism isdisrupted in bladder cancer. Recognition of alterations in this novelmechanism may have biological and clinical implications that deservefurther analyses.

ACKNOWLEDGMENTS

We thank Drs. William Gerald and Andrew Koff for their suggestions andcritical review of the manuscript.

Fig. 5. Proposed model of intranuclear compartmentalization of cyclin E during cellcycle progression. Cells have undetectable levels of cyclin E during G0 and early G1phases. They express cyclin E with a nucleoplasmic pattern during mid-G1 phase, whereascyclin E localizes to nucleoli during the G1-S transition. Finally, cyclin E is undetectableduring G2-M phase. Dynamic changes in cyclin E affect not only the expression levels butintracellular localization as well. Shuttling of cyclin E from the nucleus to the nucleolusand finally to the cytoplasm might serve as a titration mechanism, presenting cyclin E forenzymatic degradation and thus allowing progression through the cell cycle.

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