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doi: 10.1152/ajprenal.00427.2011302:F905-F916, 2012. First published 18 January 2012;Am J Physiol Renal Physiol

Gmuender, Joost Van Delft, Michael P. Ryan and Tara McMorrowRobert Radford, Craig Slattery, Paul Jennings, Oliver Blaque, Walter Pfaller, Hanseffects on the cell cyclerenal proximal tubular epithelial cells independently of Carcinogens induce loss of the primary cilium in human

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CALL FOR PAPERS Biology of the Central Cilium and Cystic Diseases

of the Kidney

Carcinogens induce loss of the primary cilium in human renal proximaltubular epithelial cells independently of effects on the cell cycle

Robert Radford,1 Craig Slattery,1 Paul Jennings,2 Oliver Blaque,1 Walter Pfaller,2 Hans Gmuender,3

Joost Van Delft,4 Michael P. Ryan,1 and Tara McMorrow1

1UCD School of Biomolecular and Biomedical Research, UCD Conway Institute, University College Dublin, Dublin, Ireland;2Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria;3Genedata, Basel, Switzerland; and 4Netherlands Toxicogenomics Centre (NTC), Department of Human Biology, Departmentof Toxicogenomics, Maastricht University, Maastricht, The Netherlands

Submitted 1 August 2011; accepted in final form 13 January 2012

Radford R, Slattery C, Jennings P, Blaque O, Pfaller W,Gmuender H, Van Delft J, Ryan MP, McMorrow T. Carcinogensinduce loss of the primary cilium in human renal proximal tubularepithelial cells independently of effects on the cell cycle. Am J PhysiolRenal Physiol 302: F905–F916, 2012. First published January 18,2011; doi:10.1152/ajprenal.00427.2011.—The primary cilium is animmotile sensory and signaling organelle found on the majority ofmammalian cell types. Of the multitude of roles that the primarycilium performs, perhaps some of the most important include main-tenance of differentiation, quiescence, and cellular polarity. Given thatthe progression of cancer requires disruption of all of these processes,we have investigated the effects of several carcinogens on the primarycilium of the RPTEC/TERT1 human proximal tubular epithelial cellline. Using both scanning electron microscopy and immunofluores-cent labeling of the ciliary markers acetylated tubulin and Arl13b, weconfirmed that RPTEC/TERT1 cells express primary cilium uponreaching confluence. Treatment with the carcinogens ochratoxin A(OTA) and potassium bromate (KBrO3) caused a significant reductionin the number of ciliated cells, while exposure to nifedipine, anoncarcinogenic renal toxin, had no effect on primary cilium expres-sion. Flow cytometric analysis of the effects of all three compoundson the cell cycle revealed that only KBrO3 resulted in an increase inthe proportion of cells entering the cell cycle. Microarray analysisrevealed dysregulation of multiple pathways affecting ciliogenesisand ciliary maintenance following OTA and KBrO3 exposure, whichwere unaffected by nifedipine exposure. The primary cilium repre-sents a unique physical checkpoint with relevance to carcinogenesis.We have shown that the renal carcinogens OTA and KBrO3 causesignificant deciliation in a model of the proximal tubule. With KBrO3,this was followed by reentry into the cell cycle; however, deciliationwas not found to be associated with reentry into the cell cyclefollowing OTA exposure. Transcriptomic analysis identified dysregu-lation of Wnt signaling and ciliary trafficking in response to OTA andKBrO3 exposure.

deciliation; carcinogenesis; proximal tubule; cilia

THE PRIMARY CILIUM (also referred to as the central cilium) is animmotile, microtubule-based organelle found on the majority

of mammalian cell types. Until relatively recently, the primarycilium was considered a vestigial organelle from our unicellu-lar evolutionary lineage. However, it is now widely acknowl-edged that cilia play a central role in an increasing number ofdiverse cellular processes, including chemo- and mechanosen-sation, signal transduction, phototransduction, and develop-mental patterning (4, 5, 32, 42).

The ciliary body (or axoneme) is composed of nine microtubuledoublets enveloped in a ciliary membrane. These doublets arecomposed of �-tubulin and �-tubulin and are heavily posttrans-lationally modified through acetylation, glutamylation, or glycy-lation to increase microtubule stability and functionality (15, 29,31). The ciliary membrane forms a distinct region of the cellmembrane as lateral diffusion between it and the wider cellmembrane does not readily occur (47, 33). This results in local-ization of a distinct pool of proteins in the ciliary membranefacilitating concentration of particular receptors and signalingproteins within the cilium (34). Therefore, the primary ciliumrepresents a highly specialized sensory and signal transductionhub. Numerous receptor signaling components are highly en-riched in the cilium, including Sonic Hedgehog signaling proteins,canonical and noncanonical Wnt signaling components, the plate-let derived growth factor (PDGF) receptor (12, 26, 42), andreceptors involved in phototransduction and olfaction (3, 24). Therenal primary cilium extends into the lumen of the renal tubuleand is proposed to play a major sensory role (46).The ability ofthis organelle to sense fluid flow and initiate calcium-based signalingis thought to contribute to the maintenance of normal epithelialphenotype and function throughout the renal tubule (39, 40).

The physiological importance of the central cilium is high-lighted by the growing list of disorders where the primaryetiology is rooted in abnormal ciliary function, termed “cil-iopathies” (18). Autosomal dominant polycystic kidney disease(ADPKD), the first disease identified as a ciliopathy, is theresult of a single mutation in either polycystin-1 or polycys-tin-2. ADPKD is characterized by epithelial dedifferentiationand overproliferation, leading to progressive cyst formation,gross architectural disruption of the kidney, end-stage renaldisease, and increased risk of renal cancer (17). Other ciliopa-thies include Bardet-Beidl syndrome, situs inversus, Meckel-Grüber syndrome, and nephronophthisis (2, 6, 9, 36, 48).

Address for reprint requests and other correspondence: T. McMorrow, RenalDisease Research Group, School of Biomolecular and Biomedical Science,UCD Conway Institute, Univ. College Dublin, Dublin 4, Ireland (e-mail:[email protected]).

Am J Physiol Renal Physiol 302: F905–F916, 2012.First published January 18, 2011; doi:10.1152/ajprenal.00427.2011.

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More recently, there has been focus on the potential impor-tance of the primary cilium in carcinogenesis. Several studieshave observed that primary cilia are suspiciously absent in avariety of cancer cell types, including mammary and pancreaticcancers (43, 50). In addition, several cilia-related genes aredysregulated in cancer. For example, somatic mutations in thetumor suppressor von Hippel-Lindau, which is necessary forciliogenesis (13), occur in the majority of clear cell renal cellcarcinomas (41). In ovarian cancer cells, inhibition of aurora Akinase, which regulates cilia assembly, induced cell cyclearrest (7). However, the suggested link between the primarycilium and tumorigenesis lies in the fact that cilium formationis closely linked to the cell cycle. In proliferating cells, thecentrosomes coordinate spindle pole formation during mitosis.In quiescent or interphase (G1 phase) cells, the centrosomesmigrate to the cell surface to form the mother centriole andsubsequently the basal body, which nucleates primary ciliumformation. Therefore, an inverse relationship exists betweenprimary cilium formation and cellular proliferation. Since theprimary cilium is now known to play a role in maintainingthese cell features (14, 22), there is significant potential for theinvolvement of ciliary dysfunction in carcinogenesis.

In this study, the effects of known renal carcinogens ochra-toxin A (OTA) and potassium bromate (KBrO3) and a noncar-cinogen, nifedipine, were examined in a novel proximal tubularepithelial cell line (RPTEC/TERT1). Given the associationbetween ciliary disruption and cancer, we examined the effectsof these renal chemical carcinogens on cilia formation inproximal tubular epithelial cells.

MATERIALS AND METHODS

Cell culture and treatment. The human RPTEC/TERT1 renalproximal tubular epithelial cell line, purchased from Evercyte, wasused throughout this study. The RPTEC/TERT1 cell line was estab-lished via the introduction of human telomerase (hTERT) into primaryhuman proximal tubular epithelial cells (49). RPTEC/TERT1 cellswere maintained at 37°C in a 5% CO2 humidified atmosphere inlow-glucose (5 mM) DMEM/nutrient mix F-12 supplemented withITS, EGF, hydrocortisone, L-glutamine, and penicillin/streptomycin.Cells were maintained for at least 10 days upon reaching confluencebefore treatment to allow stabilization of the monolayer (27). Nifed-ipine, OTA, and KBrO3 were obtained from Sigma-Aldrich. In allcases, DMSO to a final concentration of 0.1% (vol/vol) was used as avehicle. In all experiments, control cells were also exposed to 0.1%DMSO.

Scanning electron microscopy. Cells were cultured to confluenceon Thermanox coverslips (Nunc, Rochester, NY). Monolayers werefixed for 45 min with Karnovsky’s fixative (2% formaldehyde, 0.5%glutaraldehyde) and postfixed for 45 min with 1% OsO4 in 0.1 Msodium cacodylate buffer (pH 7.4). Specimens were dehydrated withmethanol followed by critical point drying. Specimens were finallysputter-coated with a 30- to 50-nm gold/palladium layer for observa-tion with a scanning electron microscope (JEOL jsm-25s). Imageswere processed using Adobe Photoshop.

Resazurin assay. Differentiated RPTEC/TERT1 cells were treatedwith increasing concentrations of OTA, KBrO3, and nifedipine for 72h in a 96-well format. Following exposure, culture medium wasremoved and 100 �l of 1 mg/ml resazurin in serum-free medium wasadded to each well and incubated for 120 min at 37°C. Fluorescencewas then measured using a Victor Wallac plate reader using 530-nmexcitation wavelength and 590-nm emission wavelength. Data areexpressed as a percentage of vehicle control (0.1% DMSO).

Phase-contrast microscopy. Cellular morphology was observed byphase-contrast microscopy using a JVC high-resolution digital camera(KY-F55BE) attached to a Nikon TMS phase-contrast microscope.Micrographs were processed using Adobe Photoshop.

Immunofluorescent labeling. RPTEC/TERT1 cells were culturedon eight-well chamber slides (Nunc LabTekII) for 10 days followingconfluence and then treated as indicated for 72 h. Cells were fixedwith 3.7% paraformaldehyde for 20 min at room temperature andpermeabilized with 0.2% Triton X-100 (vol/vol). Nonspecific bindingwas reduced by blocking in 0.5% (wt/vol) BSA/PBS. Acetylated�-tubulin was labeled using a mouse anti-human antibody (1:400,Sigma-Aldrich). Zonula occludens-1 (ZO-1) was labeled using arabbit anti-human antibody (1:300, Zymed). Arl13b was labeled usinga rabbit anti-human antibody (1:200, provided by Oliver Blaque).Nuclei were stained with 4,6-diamidino-2-phenylindole (1:1,000).Slides were imaged using a Zeiss LSM510 confocal microscope, andimages were processed using ImageJ.

Flow cytometry. RPTEC/TERT1 cells were seeded on six-wellculture plates. Ten days following confluence, cells were treated with300 nM OTA, 1 mM KBrO3, or 10 �M nifedipine for 72 h. Thesupernatant was removed and stored on ice. Cells were then washedwith ice-cold PBS, and trypsinized with 5% trypsin-EDTA, whichwas then neutralized with DMEM/F-12 containing 10% (vol/vol)FCS. Supernatants were added once again to the correspondingsamples and centrifuged at 1,000 rpm for 3 min to pellet the cells.Cells were resuspended to a density of 1 � 106 cells/ml in ice-coldPBS and pelleted by centrifugation at 1,000 rpm for 3 min. Cells werethen fixed with 70% ice-cold EtOH. EtOH was removed by washingwith ice cold PBS. Cells were spun down at 3,000 rpm for 5 min andresuspended in 50 ng/ml propidium iodide in EDTA/Triton X-100buffer for at least 15 min. The effects of all three compounds on cyclewere analyzed using an Accuri C6 flow cytometer and FCS Express 4.

RNA isolation and microarray hybridization. PTEC/TERT1 cellswere grown to confluence in six-well plates. Ten days postconfluence,cells were treated with 300 nM OTA, 1 mM KBrO3 or 10 �Mnifedipine for 72 h. Total RNA was extracted (n � 3 for eachcondition) using TRIzol according to the manufacturer’s instructionsand purified using RNeasy Mini Kits (Qiagen). RNA purification andquality were assessed using the Agilent 2100 Bioanalyzer to deter-mine the 28S:18S rRNA ratio. Sample preparation, hybridization,washing, staining, and scanning of the Affymetrix Human GenomeU133 Plus 2.0 GeneChip arrays were performed at the NetherlandsToxicogenomics Centre (Maastricht, The Netherlands) according tothe manufacturer’s instructions as previously described (20).

Microarray data analysis. All analyses were based on normalizedexpression values generated with Genedata Expressionist 6.2, whichafter a quality assessment step were normalized with GeneChip RMA(GC-RMA). Normalized data were further analyzed with GenedataExpressionist 6.2 and GeneSpring (GX 10.0.1), respectively. Toidentify differentially regulated genes for each treatment, at each timepoint comparisons were made against the time-matched vehicle con-trol. Differentially expressed genes were selected based on the fold-change (2-fold change or greater) and one-way ANOVA (P � 0.05).

Bioinformatic analysis. Network analysis was conducted withMetaCore Network Tools (GeneGo, San Diego, CA). Gene SetEnrichment Analysis (GSEA) was performed to determine whetherthe cilia-related gene set was enriched within chemical datasets.GSEA was performed following the developer’s protocol (45) (http://www.broad.mit.edu/gsea/).

Statistical analysis. Loss of the primary cilium following carcino-gen exposure was quantified by determining the ratio of cilia to nucleiusing multiple independent fields of view (n � 3). The ratio of ciliato nuclei was then expressed as a percentage of the control for eachtime point. Statistical analysis was performed using one-way ANOVAand a Bonferroni posttest. Statistical significance was assumed forP � 0.05. Data are shown �SE (***P � 0.001).

F906 DECILIATION OF TUBULAR CELLS AFTER CARCINOGEN EXPOSURE

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RESULTS

Differentiated human renal proximal tubular cells express aprimary cilium. Human renal proximal tubular cells (RPTEC/TERT1) were differentiated on six-well plates. RPTEC/TERT1cells formed fluid-filled domes which were distributed acrossthe monolayer, indicating that the epithelial monolayer hadformed a functional barrier (Fig. 1Ai). The presence of cilia onRPTEC/TERT1 cells was examined by both scanning electronmicroscopy (Fig. 1A, ii and iii) and indirect immunofluorescentstaining for acetylated �-tubulin (Fig. 1B). Primary cilia ex-tended above the apical surface of the RPTEC/TERT1 andwere observed in �90% of cells. A three-dimensional (3D)projection of the collapsed Z-stack confocal images in Fig. 1Bshows the spatial distribution of acetylated �-tubulin in the

cell. Cytoplasmic microtubules were also visualized. However,this cytoplasmic network was located in a different plane fromthe primary cilium [for 3D projection, see Supplementary DataFile 1 (video); supplemental material for this article is availableon the journal website]. Using ZO-1 staining to identify cellu-lar borders in conjunction with acetylated �-tubulin staining tovisualize the primary cilium, it was observed that each cellexpressed a single primary cilium (Fig. 1C, i–iii). The length ofthe primary cilium varied from cell to cell, ranging from 10 to20 �m (Fig. 1C, iv). The small GTPase, ADP-ribosylationfactor-like protein 13b (Arl13b) is a specific marker for theprimary cilium and was seen to colocalize with acetylated�-tubulin in the primary cilium in confluent RPTEC/TERT1cells (Fig. 1D, i–iii).

Fig. 1. Confluent human proximal tubular epithe-lial RPTEC/TERT1 cells express primary cilia.Ai: phase-contrast micrograph showing 10-day con-fluent RPTEC/TERT1 cells (�100). ii and iii: Mi-crographs of confluent RPTEC/TERT1 cells at dif-ferent magnifications. B: stills from 3-dimensionalprojection (see Supplementary Data File 1; video).i: High-magnification image of immunofluorescentlylabeled acetylated �-tubulin. ii: Genetic materialstained with 4,6-diamidino-2-phenylindole (DAPI).iii: Merge. Ci: immunofluorescently labeled acety-lated �-tubulin shows the presence of multiple pri-mary cilia. ii: Cellular borders are demarcated byimmunofluorescent labeling of the tight junctioncomponent zonula occludens (ZO)-1. iii: Merge.iv: Composite image of collapsed Z-scans showsspatial distribution of the cilia in relation to thecellular surface. Di: immunofluorescently labeledacetylated �-tubulin. ii: Immunofluorescently labeledArl13b. iii: Composite images showing colocaliza-tion of acetylated �-tubulin and Arl13b to the pri-mary cilium.

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Effects of nifedipine, OTA, and KBrO3 on RPTEC/TERT1viability. The effects of OTA, KBrO3 (which are classified asIARC class IIb renal carcinogens), and nifedipine (a non-carcinogenic compound) on the viability of the RPTEC/TERT1 cell line were examined using a resazurin cellviability assay to determine the concentration of each com-pound to be used for transcriptomic profiling. The aim wasto induce a limited amount of measurable toxicity across allthree compounds. Therefore, a 10% decrease in resazurinconversion was considered an appropriate concentration forall further experiments. Working concentrations were ini-tially determined in a 96-well format (Fig. 2, A–C) and thenrefined for a 6-well format. The final concentrations to be

used for further studies were 300 nM OTA, 1 mM KBrO3,and 10 �M nifedipine. By phase-contrast microscopy, it wasobserved that while these concentrations induced significantalterations in cell morphology (such as loss of fluid-filleddomes as indicated by arrows), no dramatic reduction in cellnumbers was observed (Fig. 3, D–G). Subtle alterations inthe shape and size of cells were also observed followingtreatment with OTA, KBrO3, and nifedipine compared withvehicle controls.

Renal carcinogens induced loss of the primary cilium inproximal tubular epithelial cells. The effects of nifedipine,OTA, and KBrO3 exposure on the primary cilium of theRPTEC/TERT1 cell line was assessed using immunofluores-

Fig. 2. Effects of nifedipine, ochratoxin A (OTA), andpotassium bromate (KBrO3) on RPTEC/TERT1 via-bility and morphology. The effects of nifedipine (A),OTA (B), and KBrO3 (C) on RTPEC/TERT1 viabilityas assessed by resazurin metabolism. Results are ex-pressed as percentage of control � SE; n � 3. Alsoshown is a morphological analysis of untreatedRPTEC/TERT1 cells (D) compared with the effects of10 �M nifedipine (E), 300 nM OTA (F), and 1 mMKBrO3 (G) assessed by phase-contrast microscopy.



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cent labeling of acetylated �-tubulin. This was carried out atthree time points (6, 24, and 72 h). Exposure to nifedipine,OTA, and KBrO3 for 6 h had no effect on the proportion ofciliated cells compared with vehicle controls (Fig. 3, A–D). By24 h, however, cells exposed to either OTA or KBrO3 dis-played a definite trend toward a reduction in the proportion ofcells expressing a primary cilium (although these did not reachstatistical significance) (Fig. 3, E–H). By 72 h, RPTEC/TERT1cells exposed to either OTA or KBrO3 showed a statisticallysignificant and nearly complete loss of cilia (n � 3, ***P �0.001) (Fig. 3, I–L). While a small proportion of cells did retaincilia, these appeared dramatically shortened compared withthose of vehicle controls. In contrast, exposure to nifedipinehad no statistically significant effect on the proportion of

ciliated cells even after 72 h (Fig. 3, I–L). After 72-h exposure,there appeared to be more acetylated �-tubulin in the cytosolfollowing exposure to OTA and particularly KBrO3 comparedwith vehicle controls and nifedipine-treated cells.

Effects of OTA and KBrO3 on the microtubule network. Tofurther explore the finding of increased acetylated �-tubulin inthe cytosol following OTA and KBrO3 exposure, we decidedto investigate potential effects on microtubule organization byexamining total �-tubulin and �-tubulin using immunofluores-cence. Primary cilia were observed in both control and nife-dipine-treated cells following 72-h exposure stained with �-tu-bulin (Fig. 4, A and B) and �-tubulin (Fig. 4, E and F),although the primary cilia were less distinguishable fromcytosolic staining of microtubules than in those cells stained

Fig. 3. Exposure to carcinogens induced loss of the primary cilium. RPTEC/TERT1 cells were grown in 8-well chamber slides (Nunc) and treated 10 dayspostconfluence. A–C: control RPTEC/TERT1 cells stained for acetylated �-tubulin and DAPI at 6, 24, and 72 h. D–F: 10 �M nifedipine-treated RPTEC/TERT1cells at 6, 24, and 72 h. G–I: 300 nM OTA-treated RPTEC/TERT1 cells at 6, 24, and 72 h. J–L: 1 mM KBrO3-treated RPTEC/TERT1 cells at 6, 24, and 72h. Bottom: graph showing the number of ciliated cells obtained by determining the ratio of cilia to nuclei, expressed as a percentage of its time-matched controlfor nifedipine-, OTA-, and KBrO3-treated cells at 6, 24, and 72 h; n � 3. ***P � 0.001.



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with acetylated �-tubulin (Fig. 3, I and J). While some minoralterations in �-tubulin were observed following nifedipinetreatment (Fig. 4B), there were no gross changes in the overallmicrotubule network, which was indicated by staining witheither �-tubulin (Fig. 4, C and D) or �-tubulin (Fig. 4, G andH) following treatment with either OTA or KBrO3. This wouldindicate that the changes detected in Fig. 3 following KBrO3

and OTA were confined to acetylated �-tubulin.Effects on the cell cycle. The effects OTA, KBrO3, and

nifedipine on the cell cycle of differentiated RPTEC/TERT1cells were analyzed by propidium iodide staining using flowcytometry. Nifedipine had no significant effect on the propor-tion of cells in the G0/G1, S, and G2/M phases of the cell cycle(Fig. 5A). Surprisingly, OTA did not significantly affect the

proportions of cells in the different phases of the cell cyclecompared with vehicle control after 72-h exposure: G0/G1

(85.48 vs. 80.56%), S (2.56 vs. 2.66%), and G2/M (11.97 vs.16.78%). These results suggested that 300 nM OTA had nosignificant effect on the RPTEC/TERT1 cell cycle (Fig. 5B). Incontrast, KBrO3 caused a significant increase in the proportionof aneuploid cells. The remaining diploid cells showed adecrease in the proportion of cells in the G0/G1 phase of thecell cycle (from 85.48 to 61.15%; see Table 1), with a corre-sponding increase in the proportion of cells in G2 phase of thecell cycle (from 11.97 to 38.5%; see Table 1). These resultsindicate that the exposure to KBrO3 resulted in gross chromo-somal abnormalities with a significant proportion of diploidcells reentering the cell cycle (Fig. 5C).

Effects of nifedipine, OTA, and KBrO3 on the RPTEC/TERT1 gene expression profile. RNA was isolated from cellsexposed to 10 �M nifedipine, 300 nM OTA, and 1 mM KBrO3

at 6, 24, and 72 h. Gene expression profiles at each time pointwere compared with time-matched vehicle controls to identifydifferentially regulated genes. At 6 h, 31 genes were signifi-cantly affected by nifedipine (24 downregulated and 7 upregu-lated), 7,564 genes were significantly affected by OTA (5,260downregulated and 2,304 upregulated), and 148 genes weresignificantly affected by KBrO3 (90 downregulated and 58upregulated). At 24 h, 375 genes were significantly affected bynifedipine (365 downregulated and 10 upregulated), 10,802genes were significantly affected by OTA (6,902 downregu-lated and 3,900 upregulated), and 1,736 genes were signifi-cantly affected by KBrO3 (1,347 downregulated and 389 up-regulated). By 72 h, 136 genes were significantly affected bynifedipine (63 downregulated and 73 upregulated), 6,944 geneswere significantly affected by OTA (3,802 downregulated and3,142 upregulated), and 3,149 genes were significantly affectedby KBrO3 (1,951 downregulated and 1,198 upregulated).

Cilia gene set. Using a number of publicly available sourcesincluding the Ciliome database (http://www.sfu.ca/leroux/ciliome_database.htm) and published literature, a list of geneswhich are known to be involved in ciliogenesis and mainte-nance of the cilium was compiled to form a cilia gene set (seeSupplementary Table S3 for the full cilia gene set). GSEA wasperformed on the differentially regulated data sets for eachcompound to determine whether the cilia gene set was enrichedin each condition (Fig. 6). The cilia gene set was not found tobe enriched at any time point in cells exposed to nifedipine. Incontrast, the cilia gene set was enriched after OTA treatment atall three time points with the highest enrichment score ob-served after 6 h. The cilia gene set was also enriched byexposure to KBrO3 at the 24- and 72-h time points, but not at6 h. This analysis suggests that significant numbers of genesrelated to cilia formation and maintenance were dysregulatedby exposure to OTA and KBrO3. To further characterize thenature of these transcriptomic changes, a detailed characteriza-tion of the individual genes affected was performed and issummarized in Table 2. Nifedipine did not result in dysregu-lation of any ciliary genes at any time point in the RPTEC/TERT1 cell line. Of the genes found to be significantly dys-regulated by OTA and KBrO3, genes involved in ciliary tar-geting such as KIF3A, KIF3B, ARL6, and several of the BBSgene family members were dysregulated. Of note was theobservation that the vast majority of the genes which weredysregulated by OTA or KBrO3 were downregulated.

Fig. 4. Effects of OTA and KBrO3 on the tubulin microtubule network.RPTEC/TERT1 cells were grown in 8-well chamber slides (Nunc) and treated10 days postconfluence. Shown are control RPTEC/TERT1 cells (A) and cellsstained for �-tubulin following 72 h exposure to 10 �M nifedipine (B), 300 nMOTA (C), and 1 mM KBrO3 (D). Also shown are control RPTEC/TERT1 cells(E) and cells stained for �-tubulin at 72 h, 10 �M nifedipine (F), 300 nM OTA(G), and 1 mM KBrO3 (H).



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To further characterize the effects of OTA and KBrO3 oncanonical signaling pathways in RPTEC/TERT1 cells, a globalpathway analysis of the transcriptomic data was performedusing MetaCore (Table 3). This analysis identified the mostsignificantly dysregulated pathways in OTA- and KBrO3-treated cells. A number of pathways with relevance to cancerdevelopment were identified, and several of these such as Wnt,transforming growth factor (TGF)-� related cytoskeletal re-modeling, cell cycle, epithelial-mesenchymal transition, andcell adhesion were common to both OTA and KBrO3 (Table3). However, in some cases, OTA and KBrO3 resulted inopposing effects on these common pathways. For example, thecell cycle influence of Ras and Rho proteins on the G1/Stransition network was negatively regulated by OTA, but thisgene network was positively regulated by KBrO3.

DISCUSSION

The primary cilium is essential for a wide variety of cellularfunctions (8, 44). The ubiquitous presence of this organelleindicates that the primary cilium likely plays an important roleacross many cell types and species. In this study, we haveshown that differentiated human proximal tubular epithelialcells express a single primary cilium. Furthermore, we haveshown that after exposure to two renal carcinogens these cellslose their primary cilium. This deciliation was not a general-ized response to nephrotoxin exposure, and perhaps moresignificantly, loss of the primary cilium did not necessarilycoincide with reentry into the cell cycle. While KBrO3 inducedsignificant chromosomal abnormalities and limited reentry intothe cell cycle, OTA had no effect on the cell cycle, suggestingthat there are likely diverse mechanisms underlying the effectsof these two carcinogens. Gene expression analysis demon-strated that loss of the cilium was correlated with significantdysregulation of cilia-related genes and further underlined thedivergent mechanisms of OTA- and KBrO3-induced decilia-tion.

Loss of the primary cilium may occur through two distinctmechanisms. The cilium may be reabsorbed into the cell body,or the entire organelle may be shed from the surface of the cell.Removal of the primary cilium through reabsorption occurs ascells progress through the cell cycle. Indeed, removal of thecilium is a necessary event for mitosis to occur as the mothercentrioles which nucleate the primary cilia also contribute toformation of the centrosome, the microtubule-organizing cen-ter responsible for arranging the mitotic spindles during mitosis(23). Ciliary shedding, on the other hand, may be a response toexposure to toxic stimuli (37). In this study, we have shownthat loss of the primary cilium occurred following exposure ofhuman renal proximal tubular cells to the renal carcinogensKBrO3 and OTA as early as 24 h following exposure, and thatby 72 h deciliation was almost complete. The loss of the ciliawas also associated with increased levels of acetylated �-tu-bulin in the cytosol, especially following treatment with

Fig. 5. Alteration of the cell cycle of confluent RPTEC/TERT1 cells. RPTEC/TERT1 cells were treated 10 days postconfluence with 300 nM OTA or 1 mMKBrO3 for 72 h. The effects on the cell cycle as measured by flow cytometricanalysis of propidium iodide staining are shown for 10 �M nifedipine (A), 300nM OTA (B), and 1 mM KBrO3 (C) treatment (n � 3). Vehicle control resultsare shown in black, and treated cell results are shown in red.



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KBrO3. These findings are in agreement with a study onchemical deciliation in the Madin-Darby canine kidney tubularcell line, which reported an increase in the cytosolic staining ofacetylated �-tubulin (37). The authors of that study interpretedtheir findings as indicating that cells continue to acetylate�-tubulin molecules, even during stress-induced deciliation,

but that this pool of acetylated tubulin remains cytosolic asthere is no ciliary axoneme to incorporate it. In our presentstudy, the primary cilia were unaffected by the noncarcino-genic nifedipine, so this was not a generalized response to thepresence of a chemical stressor. However, a wider panel ofchemical carcinogens should be examined in future studies todetermine whether this effect is an important event duringchemical carcinogenesis or a compound-specific observation.

To further characterize the mechanisms governing loss ofthe primary cilium following exposure to both OTA andKBrO3, we analyzed the effects of both compounds on thegene expression profile of the RPTEC/TERT1 cells at early (6h), medium (24 h), and late (72 h) time points. A gene set withknown ciliary function was compiled to facilitate a targetedapproach to this analysis. Both OTA and KBrO3 were found tocause significant dysregulation of many genes involved inciliogenesis and cilia maintenance in RPTEC/TERT1 cells.Pathway analysis of the RPTEC/TERT1 transcriptomic datausing MetaCore pathway analysis revealed that Wnt signalingand cytoskeletal remodeling were ranked the most significantlyaltered pathway following both OTA and KBrO3 treatment.Previous studies have shown that loss of the primary ciliumresults in enhanced canonical Wnt signaling in cells (16).Recent evidence has suggested that �-catenin is sequestered inthe cilium, suggesting that an intact cilium acts as a negativeregulator of Wnt signaling (25). Since canonical Wnt signalingis implicated in the progression of malignant tumors, this is apotential mechanistic link between deciliation and carcinogen-esis. Investigation of individual cilia-related genes revealed anumber of potentially important transcriptional alterations.Kinesin II is a heterotrimeric motor complex composed ofKif3a, Kif3b, and KAP-3, which is responsible for anterogradetransport to the ciliary tip. Mice lacking correct kinesin IIfunction fail to form primary cilia (28). In the current study,OTA caused downregulation KIF3A as early as 6 h, and thisdownregulation was maintained until the 24-h time point whenboth of the remaining components of kinesin II, KIF3B andKIFAP3, were also downregulated, suggesting significant dis-ruption of anterograde transport following OTA exposure. Atthe medium and late time points, numerous components of theintraflagellar transport or IFT pathway and BBS genes weredysregulated. Given that BBS and IFT pathways are known tobe involved in maintenance of the primary cilium, this findingsupports the time-dependent effects on deciliation observedfollowing OTA and KBrO3 exposure at the medium and latetime points. Anterograde IFT is responsible for transport ofintracellular cargo, including receptors, signaling components,and tubulin, to the cilium. The BBSome is believed to play arole in targeting membrane proteins to the primary cilium (21),and knockdown of individual components in either pathway

Table 1. Distribution of cell populations residing in G1, G2, and S phase of the cell cycle

G1 Mean G1 CV %G1 G2 Mean G2 CV %G2 %S G2/G1

Control 15,640.63 9.12 85.48 30,551.4 8.87 11.97 2.56 1.95Nifedipine 18,353.33 7.48 93.83 33,320.14 6.19 4.32 1.85 1.82OTA 15,622.12 10.11 80.56 30,787.1 9.03 16.78 2.66 1.97KBrO3 11,876.28 6.85 61.15 21,456.56 6.88 38.85 1.81 1.81

Percentage of the cell populations residing within each phase of the cell cycle following nifedipine, ochratoxin A (OTA), and potassium bromate (KBrO3)treatment for 72 h.

Fig. 6. Effects of carcinogens on cilia-related genes. Gene Set EnrichmentAnalysis (GSEA) was performed to determine if the cilia gene set wassignificantly enriched in nifedipine-, OTA-, and KBrO3-treated cells. A sampleGSEA enrichment plot is depicted.



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has been shown to hinder construction of the primary cilium(28, 35, 38). Our data indicate that OTA and KBrO3 mayinhibit anterograde transport toward the cilium, resulting inshortening and ultimately reabsorption of the cilium. Theretrograde motor dynein 2, which is responsible for transportfrom the cilium toward the cell, was also downregulated byOTA at all three time points and by KBrO3 at 24 and 72 h.Studies have shown that dynein 2 depletion can result inshortening of the cilium (1, 19, 30).

Since loss of the primary cilium facilitates progressionthrough the cell cycle, we examined whether OTA or KBrO3

caused alterations in the cell cycle. Nifedipine, which did notcause loss of primary cilia, did not significantly affect the

proportion of cells residing in the G0/G1 phase of the cell cycle.Similarly, exposure to OTA did not induce an observable effecton the cell cycle. Previous studies have reported that OTAinduces mitotic arrest (11), and downregulation of several celldivision cycle proteins including Cdc25 and Cdk1 was seen 24h following OTA treatment of GES-1 cells (10). In the currentstudy, pathway analysis of transcriptomic data suggested thatOTA may negatively regulate factors which promote G2/Sprogression. In contrast, KBrO3 caused a significant increase inthe proportion of cells in the G2/S phase, and this finding wasin keeping with our pathway analysis which identified the cellcycle as the sixth most significantly dysregulated pathwayfollowing exposure to KBrO3. It is unclear whether loss of the

Table 2. Effects of carcinogens on cilia-related genes

6 h 24 h 72 h

Gene Symbol Gene Name OTA KBrO3 Nif OTA KBrO3 Nif OTA KBrO3 Nif

ADCY7 Adenylate cyclase 7 2.13ALMS1 Alstrom syndrome 1 24.96 4.19 2.11AHI1 Abelson helper integration site 1 2.16ARL3 ADP-ribosylation factor-like 3 3.44 2.66 7.67ARL6 ADP-ribosylation factor-like protein 6 8.71 5.45 2.55 2.4ARL13B ADP-ribosylation factor-like protein 13b 12.23B2M �2- Microglobulin 2.33BBS1 Bardet-Biedl syndrome 1 10.35 4.9BBS2 Bardet-Biedl syndrome 2 15.03 2.02 2.89 3BBS4 Bardet-Biedl syndrome 4 2.39 6.94BBS7 Bardet-Biedl syndrome 7 6.07 11.31 9.48BBS9 Bardet-Biedl syndrome 9 2.38 2.57 2.27CALM1 Calmodulin 1 2.04CDC42 Cell division control 42 3.64CETN2 Centrin 2 2.23CEP290 Centrosomal protein of 290 kDa 2.23CLUAP1 Clusterin-associated protein 1 9.34 15.56 4.03 2.58DNAJB13 DNAJB13 2.23 2.04 2.01 2.04 2.01DYNC2H1 Dynein 2 heavy chain 1 2.25 3.48DYNC2L1 Dynein 2 light intermediate chain 1 3.69 4.43 2.73 4.78 3.36FAT4 FAT tumor suppressor homolog 4 2.5ICQB1 IQ calmodulin-binding motif-containing 1 5.26IFT57 Intraflagellar transport 57 2.47 2.47 6.76 3IFT80 Intraflagellar transport 80 8.01 2.77 3.46IFT81 Intraflagellar transport 81 11.4 2.9 3.27 3.64IFT88 Intraflagellar transport 88 2.11 2.02IFT122 Intraflagellar transport 122 2.15 2.25 2.15 2.56IFT172 Intraflagellar transport 172 4.88 2.84INVS Inversin 2.12 2.27INTU Inturned 3.95KIFAP3 Kinesin-associated protein 3 2.99KIF3A Kinesin family member 3A 5.34 2.48KIF3B Kinesin family member 3B 2.19MAPK1 Mitogen- activated protein kinase 1 2.26 4.67 2.14 2.29 2.13 3.51MAPRE1 Microtubule-associated protein RP/EB family 1 2.71MKKS McKusick-Kaufman syndrome 5.11NPHP1 Nephronophthisis 1 2.07 3.2 2.17 3.2 2.17OFD1 Oral-facial-digital syndrome 1 15.06PKD1 Polycystin-1 2.74 4.81PTCH1 Patched 1 5.85 –RPL13A Ribosomal protein L13a 2.23RSPH1 Radial spoke head 1 3.26RSPH10b Radial spoke head 10b 2.16SHH Sonic Hedgehog 2.3STAT6 Signal transduction and transcription 6 2.64TSC1 Tuberous sclerosis 1 2.42TSC2 Tuberous sclerosis 2 2.34 2.27TUBA1A �-Tubulin 1a 5.65 3.87

Fold-change of cilia-related genes found to be significantly dysregulated by 300 nM OTA or 1 mM KBrO3. Nif, nifedipine. P � 0.05, fold-change �2 at 6-,24-, and 72-h time points.



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primary cilia is a cause of, or a result of, cells reentering thecell cycle following treatment. However, our observation thatOTA-induced deciliation appears to occur independently ofcell cycle progression is a significant finding and one thatrequires further investigation. Taken together, these resultssuggest that while both OTA and KBrO3 induced deciliation inthe RPTEC/TERT1 cells, distinct mechanisms are likely in-volved, and the divergent effects on cell cycle may be relatedto the proposed mechanisms of carcinogenicity for each chem-ical (i.e., OTA is a nongenotoxic, promoting agent, whileKBrO3 is directly genotoxic).

The current study has characterized the primary cilium in anovel human renal proximal tubular epithelial cell line,RPTEC/TERT1. We have identified loss of the primary ciliumas a potentially important effect of exposure to chemicalcarcinogens, and further elucidation of the mechanisms under-

lying these deciliation events will further enhance our under-standing of the role of the primary cilium in carcinogenesis.

ACKNOWLEDGMENTS

The authors acknowledge the assistance of Dr. Alfonso Blanco Fernandezin the Flow Cytometry Core Technology Facility at the UCD Conway Institute.

GRANTS

This work was funded by the EU 6th Framework grant “carcino-GENOMICS”; PL-037712. The Conway Institute of Biomolecular and Biomed-ical Research is supported under the Program for Research in Third LevelInstitutions administered by the Higher Education Authority of Ireland. C. Slatteryis a Government of Ireland Research Fellow supported by the Irish ResearchCouncil for Science, Technology and Engineering.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

Table 3. Pathway analysis of significantly dysregulated pathways following OTA and KBrO3 treatment

Rank OTA Maps P Value KBrO3 Maps P Value

1 Cytoskeleton remodeling_TGF, WNT andcytoskeletal remodeling

6.718E-11 Cytoskeleton remodeling_TGF, WNT andcytoskeletal remodeling

6.718E-11

2 Cytoskeleton remodeling_Cytoskeleton remodeling 2.512E-10 Cytoskeleton remodeling_Cytoskeleton remodeling 2.512E-103 Translation_Non-genomic (rapid) action of

Androgen Receptor1.276E-08 Cell adhesion_Chemokines and adhesion 9.296E-08

4 Development_TGF-�-dependent induction of EMTvia MAPK

6.784E-08 Development_ERBB-family signaling 2.456E-07

5 Cell adhesion_Chemokines and adhesion 9.296E-08 Development_EGFR signaling pathway 6.862E-076 Cell cycle_Influence of Ras and Rho proteins on

G1/S Transition8.018E-07 Cell cycle_Influence of Ras and Rho proteins on

G1/S Transition8.018E-07

7 Neurophysiological process_Receptor-mediatedaxon growth repulsion

8.424E-07 Development_TGF-�-dependent induction of EMTvia RhoA, PI3K and ILK.

2.076E-06

8 Development_Growth hormone signaling viaPI3K/AKT and MAPK cascades

1.258E-06 Translation_Non-genomic (rapid) action ofAndrogen Receptor

1.276E-08

9 Signal transduction_PTEN pathway 1.265E-06 Development_TGF-�-dependent induction of EMTvia MAPK

6.784E-08

10 G-protein signaling_Regulation of p38 and JNKsignaling mediated by G-proteins

1.866E-06 PGE2 pathways in cancer 3.327E-06

11 Development_EGFR signaling pathway 6.862E-07 Neurophysiological process_Receptor-mediatedaxon growth repulsion

8.424E-07

12 Development_TGF-�-dependent induction of EMTvia RhoA, PI3K and ILK.

2.076E-06 Signal transduction_PTEN pathway 1.265E-06

13 Development_Regulation of epithelial-to-mesenchymal transition (EMT)

7.100E-06 Transcription_P53 signaling pathway 1.305E-05

14 Development_IGF-1 receptor signaling 7.844E-06 Development_Regulation of epithelial-to-mesenchymal transition (EMT)

7.100E-06

15 Cell adhesion_Plasmin signaling 9.750E-06 Signal transduction_AKT signaling 3.587E-0516 Cell adhesion_Integrin-mediated cell adhesion and

migration1.221E-05 Reproduction_GnRH signaling 1.761E-05

17 SCAP/SREBP Transcriptional Control ofCholesterol and FA Biosynthesis

1.430E-05 Cytoskeleton remodeling_Integrin outside-insignaling

1.303E-04

18 Reproduction_GnRH signaling 1.761E-05 Development_Growth hormone signaling viaPI3K/AKT and MAPK cascades

1.258E-06

19 Proteolysis_Putative SUMO-1 pathway 2.139E-05 Development_TGF-�-dependent induction of EMTvia SMADs

2.091E-05

20 Muscle contraction_Regulation of eNOS activityin endothelial cells

2.572E-05 Development_TGF-� receptor signaling 1.581E-04

21 Development_Endothelin-1/EDNRAtransactivation of EGFR

2.656E-05 Development_IGF-1 receptor signaling 7.844E-06

22 Signal transduction_Erk Interactions:Inhibition of Erk

3.342E-05 Immune response_Oncostatin M signaling viaMAPK in human cells

1.011E-04

23 Signal transduction_AKT signaling 3.587E-05 Stellate cells activation and liver fibrosis 1.415E-0424 Development_ERBB-family signaling 2.456E-07 Regulation of lipid metabolism_Regulation of

lipid metabolism via LXR, NF-Y and SREBP3.472E-04

25 Development_TGF-�-dependent induction of EMTvia SMADs

2.091E-05 Cytoskeleton remodeling_Fibronectin-bindingintegrins in cell motility

2.461E-04

The most significantly dysregulated canonical pathways as determined by MetaCore pathway analysis for both OTA and KBrO3 are listed. Those pathwayswhich are unique to either OTA or KBrO3 treatment are shown in bold, while the remaining pathways are common to both treatment groups.



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AUTHOR CONTRIBUTIONS

Author contributions: R.R., C.S., O.B., M.P.R., and T.M. provided concep-tion and design of research; R.R., C.S., and P.J. performed experiments; R.R.,C.S., H.G., J.V.D., and T.M. analyzed data; R.R., C.S., O.B., W.P., and M.P.R.interpreted results of experiments; R.R. and C.S. prepared figures; R.R. andC.S. drafted manuscript; P.J., O.B., W.P., H.G., J.V.D., M.P.R., and T.M.edited and revised manuscript; T.M. approved final version of manuscript.

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