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Involvement of cyclin B1 in progesterone-mediated cell growth inhibition, G2/M cell cycle arrest, and… Tang, Li; Zhang, Yu; Pan, Hong; Luo, Qiong; Zhu, Xiao-Ming; Dong, Min-Yue; Leung, Peter C; Sheng, Jian-Zhong; Huang, He-Feng Dec 7, 2009

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ralReproductive Biology and ssBioMed CentEndocrinologyOpen AcceResearchInvolvement of cyclin B1 in progesterone-mediated cell growth inhibition, G2/M cell cycle arrest, and apoptosis in human endometrial cellLi Tang†1,4, Yu Zhang†1, Hong Pan1, Qiong Luo1, Xiao-Ming Zhu1, Min-Yue Dong1, Peter CK Leung3, Jian-Zhong Sheng*2 and He-Feng Huang*1Address: 1Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China, 2Department of Pathology and Pathophysiology, School of Medicine, Zhejiang University, Hangzhou, China, 3Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V6H 3V5, Canada and 4Department of Reproductive Endocrinology, The First People's Hospital of Yunnan Province, Yunnan,  ChinaEmail: Li Tang - tanglikmkh@yahoo.cn; Yu Zhang - zhangyu_zy@hotmail.com; Hong Pan - panhong1978@tom.com; Qiong Luo - luoqionglq@hotmail.com; Xiao-Ming Zhu - ntzhuxm@hotmail.com; Min-Yue Dong - mydong.cn@hotmail.com; Peter CK Leung - peleung@interchange.ubc.ca; Jian-Zhong Sheng* - shengjz@zju.edu.cn; He-Feng Huang* - huanghefg@hotmail.com* Corresponding authors    †Equal contributorsAbstractBackground: Progesterone plays an important role in the proliferation and differentiation ofhuman endometrial cells (hECs). Large-dose treatment with progesterone has been used fortreatment of endometrial proliferative disorders. However, the mechanisms behind remainunknown.Methods: To investigate the role of cyclin B1 in proliferation and differentiation of hECs inmenstrual cycle, the expression of cyclin B1 throughout the menstrual cycle was evaluated in hECs.To determine the effects of progesterone on the proliferation, cell cycle progression and apoptosisof hECs and to test if cyclin B1 is involved in these effects, progesterone and/or Alsterpaullone (Alp,a specific inhibitor of Cyclin B1/Cdc2) were added to primary hECs. Cellular proliferation wasevaluated with MTT test, cell cycle with propidium iodide (PI) staining and flow cytometry,apoptosis with FITC-Annexin V and the expression of cyclin B1 with Western blotting.Results: The expression level of cyclin B1 in secretory endometria was significantly lower than inproliferative endometria (p < 0.01). Progesterone significantly inhibited the growth of hECs in aconcentration-dependent manner (P < 0.01). The treatment with progesterone significantlydecreased the expression of cyclin B1, increased the proportions of cell in G2/M phase, andapoptotic cells (P < 0.05 for all). The presence of Alp significantly enhanced the effects ofprogesterone on cyclin B1 down-regulation, G2/M cell cycle arrest and induction of apoptosis (P <0.01 for all).Conclusion: Our findings suggest that cyclin B1 is a critical factor in proliferation anddifferentiation of hECs. Progesterone may inhibit cell proliferation, mediate G2/M cell cycle arrestand induce apoptosis in hECs via down-regulating Cyclin B1.Published: 7 December 2009Reproductive Biology and Endocrinology 2009, 7:144 doi:10.1186/1477-7827-7-144Received: 16 October 2009Accepted: 7 December 2009This article is available from: http://www.rbej.com/content/7/1/144© 2009 Tang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8(page number not for citation purposes)Reproductive Biology and Endocrinology 2009, 7:144 http://www.rbej.com/content/7/1/144BackgroundProgesterone plays a pivotal role in female reproduction.It modifies the effects of estrogen on the endometrium[1].Estrogen stimulates proliferation of both glandular epi-thelial cells and stromal cells, whereas progesterone pre-vents this effect and induces secretory changes inglandular epithelial cells and decidual changes in stromalcells[2]. The balance between these two hormones playsimportant roles in regulation of the menstrual cycle, ovu-lation, implantation and pregnancy.The potent anti-proliferative effect of progesterone hasbeen utilized for treatment of endometrial proliferativedisorders[3]. Clinically, progesterone has been used forcontraception and the treatment of endometrial hyperpla-sia and adenocarcinoma as well as endometriosis [4-6]. Itis known that long term and large dose treatment withprogesterone analogs may lead to the limitation ofendometrial growth, atrophy, apoptosis and even celldeath[7]. Therapeutic use of progesterone is often associ-ated with irregular and unwanted bleeding[1]. Recentclinical studies have also raised concern about anincreased risk of cardiovascular disease or breast can-cer[8]. It highlights the importance of insights frommolecular biology of progesterone action onendometrium which may provide us with more precisemarkers for progesterone actions and thus help avoidside-effects and lead to new therapeutic proposal.Previous studies have shown that progesterone regulatesendometrial cell proliferation and differentiation througha nuclear receptor-mediated mechanism, such as down-regulation of estrogen receptor[9,10]. The progesterone-induced growth suppression of endometrial cells has alsobeen explained in various ways such as the elevated activ-ity of steroid metabolizing enzymes[11], growth factorsand cytokines[12]. However, the underlying molecularmechanisms by which progesterone negatively regulatesthe growth of endometrial cells are still not fully under-stood.Cell proliferation is restrained through the control of thecell cycle[13]. Cyclin B1 is the key component of the cellcycle machinery[14]. Cyclin B1 binds to Cdc2 at thebeginning of G2 phase forming an activated cyclin B1/Cdc2 complex and then phosphorylates its downstreamsubstrates which control the G2 to M transition and pro-mote cell mitotic division[15]. Unscheduled mis-regula-tion of cyclin B1 during the cell cycle leads touncontrolled cell growth and aberrant cell function[16]. Itis also reported that cyclins are functionally involved inthe rhythmic proliferation of normal human endometrialtissue[17]. Moreover, upregulated expression of cyclin B1,ation and differentiation[18]. Up to date, it is still unclearwhether cyclins are mediated in the negative regulation ofthe endometrium by progesterone.As the detection of significantly down-regulated expres-sion of cyclin B1 in secretory endometria strongly suggeststhat cyclin B1 plays an important role in proliferation anddifferentiation of hECs under steroids regulation, we thenexamined the effects of progesterone on the proliferation,cell cycle progression and apoptosis of hECs and tested ifcyclin B1 is involved in these effects. In addition, we deter-mined whether Alsterpaullone (Alp, a specific inhibitor ofCyclin B1/Cdc2) is capable of enhancing the effects ofprogesterone on cyclin B1 down-regulation, G2/M cellcycle arrest and induction of apoptosis.MethodsSubjectsEndometrial tissues were obtained from 18 women (atproliferative phase) who underwent hysterectomy or hys-teroscopy and 12 women (at middle secretory phase) whounderwent aspiration biopsy during IVF program forbenign uterine diseases in Women's Hospital, School ofMedicine, Zhejiang University. Written informed con-sents were obtained from all subjects and the InstitutionalReview Board of School of Medicine, Zhejiang University,granted the ethical approval for the current investigation.All the women had regular menstrual cycles and receivedno hormonal treatments three months prior to the opera-tion. Their ages ranged from 29 to 47 years. The menstrualcycle phase was confirmed by histologic dating.Cell culture6 cases of endometrial tissue at proliferative phase wereminced in Hanks' solution and digested with 0.2% colla-genase (Gibco-BRL, Gaithersburg, MD, USA) at 37°C for50 min. The dispersed cells were filtered through a 70-mmnylon mesh to remove the undigested tissue pieces. Cells,containing endometrial epithelial cells and endometrialstromal cells were collected and re-suspended in Dul-becco's modified Eagle's medium (DMEM, Gibco-BRL)supplemented with 10% fetal bovine serum (Invitrogen),50 U/ml penicillin, and 50 μg/ml streptomycin in a 60-mm or 10-mm petri dish or 24 well plate in a humidifiedatmosphere of 5% CO2 at 37°C. The medium waschanged every day. Progesterone at the concentration of 1× 10-9, 1 × 10-8, 1 × 10-7 or 1 × 10-6 M or progesterone (1 ×10-7 M) and/or Alp (5 mM) (Calbiochem, USA) wasadded and the cells were incubated for another 72 hbefore specific experiments. Each experiment wasrepeated at least three times.MTT assayPage 2 of 8(page number not for citation purposes)cyclin D1 and cyclin E was detected in endometrial carci-nomas, which indicated that cyclins might be the majorcell cycle regulators involved in endometrial cell prolifer-MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazo-lium bromide) assay was used to evaluate the cellular pro-liferation. Briefly, after hECs were treated withReproductive Biology and Endocrinology 2009, 7:144 http://www.rbej.com/content/7/1/144progesterone for 72 h, 20 μl of MTT (5 mg/ml) (Sigma,USA) was added and the cells were incubated for addi-tional 4 h at 37°C. When the incubation finished, 200 μlof Dimethyl sulfoxide (DMSO) was added and the opticaldensities (OD) were read at 490 nm with a microplatereader. The experiment was conducted in triplicate andrepeated three times. Inhibition rate was calculated as fol-lowing: (1-OD sample/OD control) × 100%.Cell cycle analysisCells were detached by trypsinization, washed three timeswith cold PBS and fixed with 80% ethanol at 4°C for 3 h.For propidium iodide (PI) staining, cells were washedthree times with PBS to remove trace ethanol. The pelletswere re-suspended and stained with propidium iodide(PI) (Sigma, USA)staining solution (0.1 mg PI and 0.5mg/ml RNase A in PBS) and incubated at 37°C for 30min. Cells were analyzed with flow cytometry FACS EPICS(Coulter Epics Altra flow cytometer; Beckman Coulter,Fullerton, CA).Detection of apoptotic cellsCells were detached, washed and re-suspended in 200 μlmedium and fluorescently labeled by addition of 20 μl ofbinding buffer and 5 μl of Annexin V-FITC (Pharmingen,SanDiego, CA). After the incubation at room temperaturein dark for 15 min, 2 μl of PI (1 mg/mL, Invitrogen, USA)was added and cells were applied to flow cytometry(Coulter Epics Altra flow cytometer). A minimum of10,000 cells with in the gated region was analyzed.Protein extraction and Western blot analysisTissues and cells were washed with PBS and lysed in lysisbuffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxy-cholate, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl flu-oride, 100 μg/ml leupeptin). The suspension wascentrifuged at 15,000 g for 15 min at 4°C, the supernatantwas collected and protein concentrations were deter-mined using the Bradford method. 30 μg of protein perlane was loaded and separated on a 10% Sodium dodecyl-sulfate (SDS)-polyacrylamide gel and transferred to Nitro-cellulose Transfer membrane (PROTRAN, BioScience,Germany). Membranes were incubated with blockingbuffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1%Tween 20 containing 5% non-fat milk) for 1 h, and thenincubated with monoclonal mouse anti-human Cyclin B1antibody (Santa Cruz Biotechnology, CA, USA, 1:200dilution), polyclonal goat anti-human β-actin antibody(Santa Cruz Biotechnology, 1:2000 dilution) in blockingbuffer overnight at 4°C. Then membranes were incubatedwith appropriate secondary antibody for 1 h at room tem-perature. The bound antibody was detected using anenhanced chemilumiscent (ECL) detection reagent (SantaCruz Biotechnology) and the bands were scanned byExpression of cyclin B1 in human endometrium detected by Western blotFigure 1Page 3 of 8(page number not for citation purposes)Expression of cyclin B1 in human endometrium detected by Western blot. The relative expression of cyclin B1 in human endometrium at the secretory phase is significantly lower than the proliferative phase (**P < 0.01).Reproductive Biology and Endocrinology 2009, 7:144 http://www.rbej.com/content/7/1/144Quantity One software (Bio-Rad Laboratories, Hercules,CA, USA). Normalized densities were determined withratio of density of cyclin B1 to that of β-actin.Statistical analysisAll data were presented as mean ± SD. Student's t-test orOne-way analysis of variance (ANOVA) were used to com-pare means. A P value less than 0.05 were considered sta-tistically significant.ResultsExpression of cyclin B1 in human endometrium12 pairs of human endometrium at proliferative phaseand middle secretory phase were subjected to Westernblot analysis. The result showed that the relative expres-sion of cyclin B1 in human endometrium at the secretoryphase is significantly lower than the proliferative phase (P< 0.01) (Figure 1).Progesterone inhibits growth of hECsWe examined the effect of progesterone on cell prolifera-tion of primary hECs. Progesterone inhibited hECsgrowth in a dose-dependent manner. Progesterone didnot inhibit hECs growth at the concentrations of 1 × 10-9M and 1 × 10-8 M but significantly inhibited cell growth atInhibition of cell growth on hECs by progesteroneFigure 2Inhibition of cell growth on hECs by progesterone. Human endometrial cells were treated with progesterone at concentrations of 1 × 10-9, 1 × 10-8, 1 × 10-7 or 1 × 10-6 M and cell growth was evaluated with MTT. Progesterone inhibited hECs growth in a dose-dependent manner. (*P < 0.05, **P < 0.01, compared with the control cells).Decreased expression of Cyclin B1 induced by progesterone and/or AlpFigure 3Decreased expression of Cyclin B1 induced by progesterone and/or Alp. Human endometrial cells were treated with progesterone and/or Alp. The expression of cyclin B1 was significantly decreased by the treatment of progesterone or proges-terone and Alp, but not Alp alone (*P < 0.05, **P < 0.01, compared with the control cells). The levels of cyclin B1 was signifi-Page 4 of 8(page number not for citation purposes)cantly decreased in cells treated with progesterone and Alp compared with those treated with progesterone or Alp alone (#P < 0.01).Reproductive Biology and Endocrinology 2009, 7:144 http://www.rbej.com/content/7/1/1441 × 10-7 M and 1 × 10-6 M with inhibitory rates of 35.0%and 70.0% respectively (P < 0.05 and P < 0.01 respectivelycompared with control cells) (Figure 2).Progesterone and/or Alp decreases the expression of cyclin B1The expression of cyclin B1 was significantly decreased bythe treatment of progesterone at a concentration of 1 × 10-7 M compared with control cells (P < 0.05), but not by thetreatment of Alp alone at a concentration of 5 mM (P >0.05). The levels of cyclin B1 was significantly decreasedin cells treated with progesterone and Alp compared withthose treated with progesterone or Alp alone or controlcells (P < 0.01 for all) (Figure 3).Progesterone or plus Alp mediate cell cycle arrest at G2/M stageThe proportion of cells in G2/M phase was significantlyterone and 19.0% after the treatment of progesterone andAlp. There was also significant difference between treat-ment with progesterone alone and progesterone plus Alp(P < 0.05) (Figure 4).Progesterone or plus Alp induce apoptosis of hECThe treatment with progesterone alone or progesteroneplus ALP significantly increased the amount of apoptoticcells (P < 0.05 and P < 0.01 respectively compared withthe control cells). The proportion of apoptotic cell was4.7% after progesterone treatment and 12.5% after treat-ment of progesterone and Alp. There was also significantdifference between treatment with progesterone aloneand progesterone plus Alp (P < 0.01) (Figure 5).DiscussionIn the present study, we demonstrated that the expressionlevel of cyclin B1 in secretory endometria was significantlyArrest of cell cycle in G2/M phase induced by progesterone and AlpFigure 4Arrest of cell cycle in G2/M phase induced by progesterone and Alp. Human endometrial cells were treated with pro-gesterone alone or in combination with Alp. Cell cycles were analyzed by flow cytometry. A: Cell cycle analyzed with propid-ium iodide (PI) staining followed by flow cytometry. B: Comparison of the proportions of cells in G2/M phase. The proportion of cells in G2/M phase was significantly increased after hECs were treated with progesterone or progesterone and Alp (*P < 0.05, **P < 0.01, compared with the control cells). There was also significant difference between treatment with progesterone alone and progesterone plus Alp (#P < 0.05).Page 5 of 8(page number not for citation purposes)increased after hECs were treated with progesterone (P <0.05), or progesterone and Alp (P < 0.01). 8.1% of cellswere arrested in G2/M phase after the treatment of proges-lower than in proliferative endometria. Progesteroneinhibited the growth of hECs in a concentration-depend-ent manner. The treatment with progesterone significantlyReproductive Biology and Endocrinology 2009, 7:144 http://www.rbej.com/content/7/1/144decreased the expression of cyclin B1, increased the pro-portions of cell in G2/M phase, and apoptotic cells. Inaddition, the presence of Alp enhanced the effects of pro-gesterone on cyclin B1 down-regulation, G2/M cell cyclearrest and induction of apoptosis. Our results indicatedthat cyclinB1 played an important role in the endometrialcell cycle progression regulated by progesterone.Cyclin B1, the master regulator in cell proliferation, playsan essential role in G2/M transition of mitosis in cell pro-liferation[19,20]. They are functionally involved in therhythmic proliferation and differentiation of humanfirmed the result of previous study that CyclinB1/Cdc2was expressed in proliferating cells of the normalendometrium, and the expression of these molecules weresuppressed in the secretory phase[17]. As the menstrualcycle is controlled by the sex steroids, mainly estrogen andprogesterone, we speculated that the down-regulatedexpression of cyclin B1 in secretory endometrium isinduced, at least partly, by the ascendant secretion of pro-gesterone.In vitro primary hECs culture confirmed the anti-prolifer-ative effect of progesterone in endometrial cell. Our dataApoptosis of hECs induced by progesterone and AlpFigure 5Apoptosis of hECs induced by progesterone and Alp. Human endometrial cells were treated with progesterone alone or in combination with Alp. Apoptosis was indexed by the detection of Annexin V. A: Apoptotic cells as labeled with Annexin V and followed by flow cytometry. B: Comparison of the proportions of apoptotic cells. The amount of apoptotic cells was sig-nificantly increased with the treatment of progesterone alone or progesterone plus ALP (*P < 0.05, **P < 0.01, compared with the control cells). There was also significant difference between treatment with progesterone alone and progesterone plus Alp (#P < 0.01).Page 6 of 8(page number not for citation purposes)endometrium and their actions are related to the levels ofsex steroids in endometrium. Our experimental data con-showed that low dose of progesterone had no evidenteffect on the proliferation of hECs, but high dose of phys-Reproductive Biology and Endocrinology 2009, 7:144 http://www.rbej.com/content/7/1/144iological level (10-7 M) [21] and an even higher level (10-6 M) could significantly inhibit the hECs proliferation. Itis consistent with clinical application of large dose proges-terone in treatment for endometrial proliferative disor-ders. As abundant expression of Cyclin B1 and alterationof the G2 pathway was reported in abnormal proliferativeendometrial tissues, such as endometrial hyperplasia,endometrial adenocarcinoma and endometrio-sis[16,18,19,22-24] the down-regulation of cyclin B1detected under the treatment of progesterone could wellexplain the underlying mechanism of the inhibitory effectof progesterone on endometrial cell growth.A few previous studies reported that the growth inhibitoryeffect of progesterone in endometrial cell was induced bythe enhancement of cell cycle arrest at the G1/Sphase[25,26]. Recently, microarray analysis of progester-one effects on endometrial cell done by Paulssen et al[27]indicated the significantly down-regulation of cyclin B1.In these studies, either mice endometrial epithelial cell orhuman endometrial epithelial cancer cell line, Ishikawacells, were used as cell model. In the current study, weused human primary endometrial cell for the treatment ofprogesterone, it was more close to the human physiology.Flow cytometry results showed that progesterone acted oncell cycle progression by regulating G2/M transition inhECs with the down-regulation of cyclin B1. We supposedthat some of the different findings were due to the differ-ent cell models and progesterone types and doses used inthe separate experiments. Results from primary humanendometrial cell culture with physiological high level pro-gesterone treatment in our experiment provided new evi-dence of the underlying mechanism.It is considered that endometrium apoptosis was regu-lated by hormonal changes[28]. In the present study,apoptosis was observed in cultured hECs and the numberof apoptotic cells was increased by the stimulation of pro-gesterone, indicating high level progesterone inducesapoptosis of endometrial cells. As G2/M arrest and apop-tosis are common phenomena after genetic damage of theG2 pathway[23], we speculated that the induction ofapoptosis in hECs by progesterone was also conducted bydown-regulation of cyclin B1. This might be one of themechanisms of anti-proliferation and endometrial atro-phy induction in women who receives large dose proges-terone treatment for endometrial proliferative disorders.The activity of cyclinB1 could be inactivated by its inhibi-tors, resulting in cell cycle arrest. CyclinB1/cdc2 inhibi-tors, such as Camptothecin and Paclitaxel, have been usedfor the treatment of malignancies and have been demon-strated to be effective in limiting tumor cell growth bytivates cyclin B1/cdc2 complex and results in the arrest ofcell growth by competitively inhibiting ATP to combinewith the catalytic domain of cdc2[29,33]. Our resultsshowed that the presence of Alp significantly enhancedthe effects of progesterone on cyclin B1 down-regulation,G2/M cell cycle arrest and induction of apoptosis. It fur-ther confirmed the pivotal role of cyclin B1 in the proges-terone active pathway in hECs. Previous studies showedthat although Alp alone induced cell arrest at G2/M phase,the proportion was only 2-fold higher than control[29].In the present study, the proportion of the G2/M cell cyclearrest induced by progesterone plus Alp was around 9-fold higher than control and 2.5-fold higher than proges-terone only. The proportion of endometrial cell apoptosisinduced by progesterone plus Alp was 12-fold higher thancontrol and 3-fold higher than progesterone only. Ourresults suggest that Alp could enhance the inhibitory effectof progesterone on endometrial cell growth and apopto-sis. Although further study is needed to clarify the mecha-nisms involved in these effects by Alp, these findingsimplicated that use of progesterone together with ingredi-ent of cell cycle inhibitors might improve the therapeuticeffect of hyperplasia, adenocarcinoma of endometriumand endometriosis. It will be interesting to validate thisconjecture in the further research.ConclusionIn conclusion, progesterone may inhibit cell proliferation,mediate G2/M cell cycle arrest and induce apoptosis inhECs via down-regulating Cyclin B1. The presence of Alpenhanced the effects of progesterone on cyclin B1 down-regulation, G2/M cell cycle arrest and induction of apop-tosis. Our findings suggest that cyclin B1 is a critical factorin proliferation and differentiation of hECs. Progesteronederivatives in combination with ingredient of CyclinB1inhibitors may be a promising way for the treatment ofendometrial proliferative diseases. Future investigationstargeting the progesterone pathway on aberrant endome-trial cell may be fruitful for developing a novel proposalof progesterone treatment.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsLT and YZ participated together with JZS and HFH in thedesign of the study. LT, YZ, HP and QL carried out theexperiments. Data analysis was performed by XMZ andLT. The manuscript was written by LT and YZ. MYD,PCKL, JZS and HFH critically read the manuscript. Allauthors read and approved the final manuscript.AcknowledgementsPage 7 of 8(page number not for citation purposes)down-regulation of cyclinB1/cdc2[29-32]. Alsterpaullone(Alp) is a specific inhibitor of cyclin B1/cdc2, which inac-We would like to give our thanks to Cai-yun Zhou for technical assistance. This work was supported by National Basic Research Program of China (No.973: 2006CB944006 and 2006CB504004).Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Reproductive Biology and Endocrinology 2009, 7:144 http://www.rbej.com/content/7/1/144References1. Druckmann R: Long-term use of progestogens--getting thebalance right: molecular biology and the endometrium.Gynecol Endocrinol 2007, 23(Suppl 1):53-61.2. 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