Activation of Sonic Hedgehog Signaling Pathway in S-type Neuroblastoma Cell Lines

J Huazhong Uni v Sci Technol ［Med Sci ］30(3):2010271Activation of Sonic Hedgehog Signaling Pathway in S-type Neuroblastoma Cell Lines *Y unan ZHOU (周昱男),Ruolian DAI (戴若连),Ling MAO (毛玲),Y uanpeng XIA (夏远鹏),Y ufang Y AO (姚玉芳),Xue Y ANG (杨雪),Bo HU (胡波)#Department of Neurology ,Union Hospital,T ongji Medical College,Huazhong University of Science and T echnology,W uhan,China 430022Huazhong University of Science and Technology and Springer-V erlag Berlin Heidelberg 2010Summary:The effects of Sonic hedgehog (Shh)signaling pathway activation on S-type neuroblastoma (NB)cell lines and its role in NB tumorigenesis were investigated.Immunohistochemistry was used to detect the expression of Shh pathway components —Patched1(PTCH1)and Gli1in 40human primary NB samples.Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1and Gli1in three kinds of S-type NB cell lines (SK-N-AS,SK-N-SH and SHEP1),re-spectively .Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway.S-type NB cell lines were treated with different concen-trations of Shh or/and cyclopamine for different durations.Cell viability was measured by using MTT method.Apoptosis rate and cell cycle were assayed by flow cytometry.The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice.High-level expres-sion of PTCH1and Gli1was detected in both NB samples and S-type NB cell lines.Cyclopamine de-creased the survival rate of the three cell lines while Shh increased it,and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment.Cyclopamine induced the cell apoptosis and the cell cycle arrest in G 0/G 1phase,while Shh induced the reverse effects and could partially pre-vent effects of cyclopamine.Cyclopamine could also inhibit the growth of NB in vivo.Our studies re-vealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle,suggesting a new therapeutic ap-proach to NB.Key words:S-type neuroblastoma cell lines;Sonic hedgehog signaling pathway;cyclopamineNeuroblastoma (NB)is a common childhood ma-lignant tumor of the sympathetic nervous system [1],aris-ing in sympathetic ganglia and adrenal medulla that are derived from neural crest stem cells [2,3].NB represents approximately 10%of childhood tumors with poor prognosis [4,5].To update,the mechanisms about the pro-liferation of NB cells are little known.Recently,many studies suggested the essential role of Sonic hedgehog (Shh)signaling pathway in regulating the development of neural crest stem [6,7],in other hand,it was approved to contribute to cell proliferation and dif-ferentiation in several human neoplasms,such as prostatecancer ,pancreas cancer and skin carcinomas [8–11].Shh,a member of the Hedgehog family of signaling molecule,is originally identified by its homology to the Drosophila melanogaster segment polarity gene hedgehog.Shh in-Yunan ZHOU,E-mail:talent62@ These author contributed equally to this work.#,@*T j y f N N S F f (N 36)duces signaling by binding to its receptor PTCH1to in-activate PTCH1and then prevents it from inhibiting the transmembrane protein Smoothened,ultimately resulting in activation of Gli.Gli,which acts as nuclear localized transcription factors,in return,regulates the expression of many target genes that control cell growth and differ-entiation in a wide variety of tissues.Aberrant activation of the Shh signaling pathway has been found to be im-plicated in the pathogenesis of various types of can-cers [12–14].There are little available data about the role of Shh signaling pathway in NB.Based on morphological ap-pearances,biochemical properties,and growth patterns,human NB cell lines can be divided into three categories:N-Type (neuroblastic),S-Type (substrate-adherent and the non-neuronal)and I-Type (the intermediate)[15].The S-type NB cells include not only malignant cells such as SK-N-AS and SK-N-SH,but also innocent cells such as SHEP1.In this study,we tried to find out the relationship between the activated Shh signaling pathway and the NB 30(3):271-277,2010J Huazhong Univ Sci Technol ［Med Sci ］DOI 10.1007/s11596-010-0342-7Corresponding author E-mail:hubo his pro ect was supported b a grant rom the ational atu-ral ciences oundation o China o.000189.pathogenesis.1MA TERIALS AND METHODS1.1Clinical Specim ensNB samples (n=40)were obtained from Union Hospital,Tongji Medical College,Huazhong University of Science and Technology (China),with approval of the Office for Scientific Research of the hospital,and all cases were reviewed by the neuropathologist to confirm the diagnosis of NB.1.2Immunohistochemistr yFor histological studies,paraffin-embedded NB samples were cut into 4-μm thick sections and stained with hematoxylin and eosin (HE).For immunohisto-chemistry,the sections were treated according to stan-dard procedures.These sections were rehydrated accord-ing to standard procedures,and treated with 10mmol/L citrate buffer (pH 6.0)at 95C for 10min to retrieve an-tigens.Following quenching of endogenous peroxidase activity with 3%H 2O 2and blocking with 10%normal goat or rabbit serum for 30min,the sections were incu-bated with goat polyclonal antibody PTCH1(1:100,Santa Cruz,USA),rabbit polyclonal antibody Gli-1(1:100,Abcam,USA)at 4°C overnight,respectively.After incubation for 16–18h,the sections were sequen-tially incubated with biotinylated rabbit anti-goat IgG ,or goat anti-rabbit IgG and the ABC reagent (V ector Labo-ratories,USA).The immunostaining results were visual-ized with 3,3’-diaminobenzidine (Sigma,USA).Finally,the sections were counter-stained with hematoxylin.Negative controls were set up in each case by replacing the primary antibody with Tris-buffered saline.1.3Cell Cult ure and Dr ug Treat mentThe S-type human NB cell lines including SK-N-AS,SK-N-SH and SHEP1were grown in Dul-becco ’s modified Eagle ’s medium (DMEM,Gibco,USA)supplemented with 10%fetal bovine serum (FBS,Gibco,USA),cultured at 37C in a humidified incubator with 5%CO 2and subcultured at the split ratio of 1:2every 3days.To detect the role of Shh signaling pathway in NB cell lines growth,Shh signaling pathway was inhibited with its selective inhibitor cyclopamine (LC Laboratories,USA),dissolved in ethanol.Exogenous Shh (R&D Sys-tems,USA)which was dissolved in phosphate-buffered saline (PBS)was used to enhance Shh signaling pathway.In the control groups,cells were treated with vehicle alone,and in the treated groups,cells were treated with different concentrations of Shh or cyclopamine for dif-ferent durations.To detect the mutual effects of Shh and cyclopamine in NB,S-type NB cells were treated with vehicle,20mol/L cyclopamine (c20),3g/mL Shh (s3),or pretreated with 3g/mL Shh for 1h,then treated with 20mol/L cyclopamine (c20+s3)for 24h,respectively.1.4RNA Extr action and Semi-quant it ative RT-PC RTotal cellular RNA was extracted using Trizol re-agent (Invitrogen,USA)according to the manufacturer ’s instructions.Total RNA (1mg)was treated with 1U/10μL Dnase Ⅰ(Life technologies,USA).Reverse transcrip-tion (RT)was carried out using a RNA polymerase chain (R)(T S z ,)T RT (L),5RN ,f y R T q R y T G RBeads (Amersham Biosciences,USA)with 0.2μmol/L of oligonucleotide primer sets.The corresponding cDNA fragments were denatured at 94C for 30s,annealed at 60C for 30s,and extended at 72C for 1min for 35cy-cles.The Oligo equences were as follows:human Gli1forward:5'-ACCCGGGGTCTCAAACTG-3';human Gli1reverse:5'-GGCTGACAGTATAGGCAGAGC-3';human Ptch1forward:5'-GACGCCGCCTTCGCTCTG-3';human Ptch1reverse:5'-GCCCACAACCAAGAAC TTGCC-3';human actin forward:5'-CCCAGCACAA TGAAGATCAA-3';human actin reverse:5'-GATCCA CACGGAGTACTTG-3'.Each RT-PCR product was analyzed by electrophoresis through a 1%agarose gel.1.5Western Blott ingCells were suspended in standard sodium dodecyl sulfate (SDS)sample buffer.Protein concentrations were determined with a Bio-Rad protein assay kit,using bo-vine serum albumin (BSA)as reference.Proteins (50ng)were separated on SDS-polyacrylamide gels electropho-resis (10%acrylamide),transferred to nitrocellulose membranes,and then probed with goat anti-PTCH1(1:200,Santa Cruz,USA),rabbit anti-Gli1(1:1000,Ab-cam,USA)or mouse anti-β-tubulin (B-5-1-2,1:2000,Sigma,USA),followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ICN,USA).Proteins were visualized with a SuperSignal West Pico chemiluminescence kit (Pierce,USA).1.6C ell Pr oliferat ion AssayCell viability was assessed using the reagent 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT,Sigma,USA)according to the manufacturer ’s protocol.Cultured cells were plated at a density of 5×103/well onto 96-well flat-bottomed tissue-culture plates,and treated with cyclopamine and/or Shh as indi-cated.At the end of the incubation period,10μL MTT (5mg/mL)was added to each well.After incubation for 4h at 37C,the cell supernatant was discarded,the MTT crystals were dissolved with DMSO,and the absorbance at 490nm (A 490)was measured by a microplate reader.Survival rate of cells was defined as the relative A of treated versus untreated controls.The experiments were repeated at least 3times.1.7Apopt osis AssayThe apoptotic rate of cells was assayed with An-nexin Ⅴ-FITC apoptosis detection kit (Biovision,USA).Briefly,after drug treatment for 24h,the cells were har-vested,washed twice with cold PBS,resuspended with 100μL binding buffer ,incubated with 5μL of Annexin Ⅴconjugated with FITC and 10μL propidium iodide (PI)for 15min at room temperature,and then analyzed by flow cytometry (Becton-Dickinson,USA).Cells in-cubated with 5μL of Annexin Ⅴconjugated with FITC and 10μL PI but without treatment were used as nega-tive controls.Annexin Ⅴ+/PI –,Annexin Ⅴ+/PI+and Annexin Ⅴ–/PI –indicated apoptotic,late apoptotic or secondarily necrotic,and normal cells,respectively.The experiments were repeated at least 3times.1.8C ell Cycle Assayy x y f y y B f y,f f ,,BS,f x 5%reaction PC kit akara hu o Japan .he prod-ucts 1m containing 0ng reverse-transcribed total A were ampli ied b Pu e a ead -o-o PC Cell c cles were e amined b low c tometr .rie l a ter drug treatment or 24h the cells were har-vested washed twice with P and i ed in 7etha-nol overnight at –20°C.Then the cells were washed once with PBS,digested with 0.2mg/mL RNase (Sigma,USA)at 70C for 20min,and stained with 0.5mg/mL PI (Sigma,USA)at room temperature for 30min.The DNA histograms were assayed with a flow cytometry by using the CELLQUEST software (Becton-Dickinson,USA).All experiments were done with 6–8wells per experi-ment and repeated at least 3times.1.9Xenogr aft Experiment5×106SK-N-AS cells were suspended in 200L of DMEM.The cell suspension was injected subcutane-ously into the both flanks of NOD/SCID female mice.Tumors were grown until they reached an average size of 200mm 3.Mice were randomly divided into two groups (n=3in each)and treated with cyclopamine (50mg kg -1day -1)or vehicle for 7days.Tumor growth was estimated by caliper measurements and tumor volumes were calculated with the formula 4/3πr 3.All animal ex-periments were approved by local ethics authorities.1.10Stat ist ical AnalysisQuantitative data were expressedas ±s.Two-tailed Student ’s t-test was performed for paired samples.Statistical analysis was performed using SPSS 12.0software (SPSS,USA).A P value <0.05was considered statistically significant.2RESULTS2.1High Expression of PTCH1and Gli1in Primar y Tumors and S-Type Hum an NB Cell LinesTo study whether the Shh signaling pathway exists in NB,we first detected the expression of PTCH1and Gli1which were the important components of Shh sig-naling pathway.Immunohistochemistry and W estern blot analysis revealed that PTCH1and Gli1were expressed highly in most of the 40primary human NB specimens and three S-type NB cell lines.PTCH1was always lo-cated on cell membrane and in cytoplasm,while Gil1clustered in nuclei and cytoplasm (fig.1A).W estern blot analysis demonstrated the proteins existence in the three cell lines (fig.1B).RT-PCR detected the PTCH1and Gli1mRNA expression in all three cell lines (fig.1C).These data indicated that Shh signaling pathway was closely associated withNB.Fig.1The expression of Shh signaling pathway components in human primary NB samples and the S-type NB cell linesA:Immunohistochemical staining for Gli1and PTCH1(as indicated by arrows)in primary human NB specimens (DAB,×400);B:Western blot analysis of the PTCH1and Gli1expression in S-type NB cell lines.All the three cell lines expressed both Gli1and PTCH1;C:RT-PCR analysis of PTCH1and Gli1mRNA levels in the S-type cell lines.All the three cell lines expressed both Gli1and PTCH1.1:SK-N-AS;2:SK-N-SH;3:SHEP12.2Effect s of Act ivated Shh Signaling Pa thway on Cell Viability of S-type NB CellsIn order to explore the effects of Shh pathway acti-vation on S-type NB cells,the Shh signaling pathway was activated or inhibited with exogenous Shh or cyclo-pamine,respectively.MTT assay was used to measure the survival rate of cells (fig.2and 3).The results y fSK-N-SH,SK-N-AS and SHEP1cells,but Shh increased the survival rate.Treatment with 3g/mL Shh and 20mol/L cyclopamine for 24h could induce maximal ef-fects.On the other hand,Shh could partially reverse the inhibition effects of cyclopamine.These findings sug-gested that activation of Shh signaling pathway was es-sential for S-type NB cells survival.showed that c clopamine decreased the survival rate oFig.2Activating Shh signaling pathway promotes the survival and growth of NB cellsA:S-type NB cell lines were treated with different concentrations of Shh for 24h.Shh increased survival rate of all the three cell lines in a dose-dependent manner,*P<0.05vs the control group treated with ethanol alone.There was no significant differ-ence between 3g/mL and 30g/mL Shh-treated groups;B:S-type NB cells were treated with 3g/mL Shh for different time durations.Shh increased survival rate of all the three cell lines early in a time-dependent manner,*P<0.05vs the control group treated with ethanol,#P<0.05vs the 4h shh-treated group.There was no significant difference between 24h and 48h shh-treatedgroups.Fig.3Blocking Shh signaling pathway inhibits the survival and growth of NB cellsA:S-type NB cells were treated with different concentrations of cyclopamine for 24h,cyclopamine decreased survival rate of all the three cell line in a dose-dependent manner,*P<0.05vs the control group,#P<0.05vs the group treated with 5mol/L cyclopamine,▲P<0.05vs the group treated with 10mol/L cyclopamine;B:After S-type NB cells were treated with 20mo/L cyclopamine for different time durations,cyclopamine decreased survival rate of all the three cell line in a time-dependent manner,*P<0.05vs the control group;C:Shh could partially reverse the inhibition effect of cyclopamine.After S-type NB cells were treated with vehicle,20mol/L cyclopamine (C20),3g/mL Shh (S3),or pretreated with 3g/mL Shh then treated with 20mol/L cyclopamine (C20+S3)for 24h,respectively,shh pretreatment partially rescued the decreased survival rates induced by cyclopamine.*P<0.05vs the control group,#P<0.05vs C20group2.3Effect s of Act ivated Shh Signaling Pa thway on Apoptosis of S-t ype NB CellsTo investigate the effects of Shh pathway on sur-vival rate of NB cell lines,flow cytometry was used to assay apoptosis.Cyclopamine increased apoptosis of S-type NB cells as compared with the control group,but shh inhibited apoptosis and reduced cyclopamine-in-duced apoptosis (fig.4).Our findings revealed that Shh y f f S y NB 2.4Effects of Activated Shh Signaling Pathway on C ell Cycle of S-t ype NB Cell LinesFlow cytometry showed the cells distributed regu-larly in G 0/G 1,S and G 2/M phases in control group,while cyclopamine induced cell cycle arrest in G 0/G 1phase,the proportion of cells in the G 0/G 1phase was increased and that in S phase decreased.Reversely,Shh induced a shift of cell cycle from G 0/G 1to S phases (fig.5).These re-S y ff f f NB y signaling pathwa took part in the regulation o apop-tosis process o -t pe cells.sults suggested that hh signaling pathwa might a ect proli eration o cells via regulating cell c cles.Fig.4Effects of Shh signaling pathway activation on apoptosis rate of S-type NB cellsS-type NB cells were treated with vehicle,20mol/L cyclopamine (C20),3g/mL Shh (S3),or pretreated with 3g/mL Shh then treated with 20mol/L cyclopamine (C20+S3)for 24h,respectively.C20could obviously induce apoptosis of all three cell lines.S3not only decreased cell apoptosis rate,but also partially reversed the effect of cyclopamine.*P<0.05vs the control group;#P<0.05vs the group treated with C20Fig.5Effects of Shh signaling pathway on cell cycle of SK-N-AS (A),SK-N-SH (B),and SHEP1(C)cells detected by using flowcytometry analysisS-type NB cells were treated with vehicle,20mol/L cyclopamine (C20),3g/mL shh (S3),or pretreated with 3g/mL Shh then treated with 20mol/L cyclopamine (C20+S3)for 24h,respectively.Cyclopamine induced cell cycle arrest in G 0/G 1phase,while Shh could partially reverse the cyclopamien-induced effects.*P<0.05vs the control group,#P<0.05vs the grouptreated with C202.4Relat ionship bet ween Shh signaling Pat hway Ac-tivation and t he Tum origenicity of NB SK-N-AS Cell LineW e examined the effect of Shh signaling pathway activation to induce tumors in immunodeficient mice.SK-N-AS NB cells were injected subcutaneously into the flanks of NOD/SCID mice.Treatment with cyclopamine (50mg kg -1day -1)resulted in the inhibition of xenograft growth and limited the increase in tumor size.During the treatment period of 7days,no adverse side effects,such as weight loss,ulcerations,or general non-well being of y (,f 6)F 6I y ff f y y fNB SK N S the animals occurred in c clopamine group or vehicle group table 1ig..ig.nhibitor e ect o c clopamine on the tumorigenicit o --A cellsTable 1Tumorigenicity of SK-N-AS cells in NOD/SCIDmice Groups Incidence Weight (g)V ehicle 6/6 1.23±0.37Cyclopamine 4/60.24±0.193DISCUSSIONOur studies first revealed that the Shh signaling pathway was widely activated in S-type human NB cell lines,which was demonstrated by high level expression of the pathway targets and components,including PTCH1and Gli1.It was further found that activation of Shh signaling pathway was critical for the growth and survival of S-type NB cells.Cyclopamine,which blocked Shh signaling pathway,led to the decreased pro-liferation in vivo and exogenous Shh partially reversed the effects.These findings suggested an essential role of Shh signaling pathway activation in the maintenance of the proliferations of S-type NB cells.The mechanisms by which the Shh signaling path-way affects the survival and proliferation of S-type NB cells are still unclear.Previous study showed that apop-tosis and cell cycle may play important roles in Shh sig-nal pathway regulating the tumorigenesis of N-type NB cells [16].In this study,we found Shh signaling pathway also affected both cell apoptosis and cell cycle in S-type NB cells.Blocking Shh signaling pathway with cyclo-pamine induced apoptosis and G 0/G 1cell cycle arrest,while activating Shh signaling pathway with Shh not only had the opposite effects but also could partially re-verse effects of cyclopamine.Bar et al found that the activated shh signaling pathway could increase anti-apoptotic protein Bcl2levels,and promote survival of medulloblastoma cells via up-regulating Bcl2[17].In the previous study,Shh signaling pathway was suggested to act as mitogen because it could promote cell cycle progression in the proliferation of neural precursor cells by maintaining the expression of G1-phase cyclins [18],which was critical to cell cycle progression from the G 1to the S phase.On the other hand,higher levels of p21and blocking S-phase entry by inactivating the cyclins expression were usually associated with the arrest of the cell cycle at G 1[19].Previous studies reported that Gli1could up-regulate the expression of some cell cycle pro-moting cyclins such as cyclinD2,FOXM1andras-ERK [20–22],and down-regulate the expression of cell cycle repressor p21in tumor cells [23].Up-regulated p21expression by cyclopamine could induce olfactory neuroblastoma cell cycle arrest at the G 1phase [24].In pancreatic tumor,Morton et al found that activated Shh signaling pathway elevated the levels of cyclin D1,and simultaneously decreased the levels of p21expression [25].W e supposed that in S-type NB maintenance and devel-opment,Shh signals might regulate the cyclins and p21,in the similar way,but detailed mechanisms involved in S-type NB still need more advanced studies.In summary,our studies suggested that the Shh signaling pathway played an important role in S-type NB cells.It could affect viability and proliferation of S-type NB y O y f NB AcknowledgmentsWe are grateful to Dr.Hanfei Ding (Cancer Center,the Medical College of Georgia)for providing not only NB cell lines,but also technical assistance.REFERENCES 1Menegaux F,Olshan AF,Neglia JP,et al.Day care,child-hood infections,and risk of neuroblastoma.Am J Epide-miol,2004,159(9):843-8512Lisewski D,Ryan S,Lim EM,et al.Concomitant compos-ite adrenal pheochromocytoma,multiple gastric stromaltumors and pseudohermaphrodism in a patient with von Recklinghausen ’s disease.Int Semin Surg Oncol,2006,3:113Dyer MA.Mouse models of childhood cancer of thenervous system.J Clin Pathol,2004,57(6):561-5764Brodeur GM.Neuroblastoma:biological insights into aclinical enigma.Nat Rev Cancer,2003,3(3):203-2165Maris JM,Matthay KK.Molecular biology of neuroblas-toma.J Clin Oncol,1999,17(7):2264-22796Ahlgren SC,Bronner-Fraser M.Inhibition of sonichedgehog signaling in vivo results in craniofacial neural crest cell death.Curr Biol,1999,9(22):1304-13147Fu M,Lui VC,Sham MH,et al.Sonic hedgehog regulatesthe proliferation,differentiation,and migration of enteric neural crest cells in gut.J Cell Biol,2004,166(5):673-6848Thayer SP,di Magliano MP,Heiser PW,et al.Hedgehogis an early and late mediator of pancreatic cancer tumori-genesis.Nature,2003,425(6960):851-8569Karhadkar SS,Bova GS,Abdallah N,et al.Hedgehogsignaling in prostate regeneration,neoplasia and metasta-sis.Nature,2004,431(7009):707-71210Couv é-Privat S,Le Bret M,Traiffort E,et al.Functionalanalysis of novel sonic hedgehog gene mutations identi-fied in basal cell carcinomas from xeroderma pigmento-sum patients.Cancer Res,2004,64(10):3559-356511Daya-Grosjean L,Couv é-Privat S.Sonic hedgehog sig-naling in basal cell carcinomas.Cancer Lett,2005,225(2):181-19212Hooper JE,Scott municating with Hedgehogs.Nat Rev Mol Cell Biol,2005,6(4):306-31713Riobo NA,Manning DR.Pathways of signal transductionemployed by vertebrate Hedgehogs.Biochem J,2007,403(3):369-37914Rubin LL,de Sauvage FJ.Targeting the Hedgehog path-way in cancer.Nat Rev Drug Discov,2006,5(12):1026-103315De Preter K,V andesompele J,Heimann P,et al.Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes.Genome Bio,2007,8(1):40116Mao L,Xia YP,Zhou YN,et al.A critical role of Sonic Hedgehog signaling in maintaining the tumorigenicity of neuroblastoma cells.Cancer Sci,2009,100(10):1848-185517Bar EE,Chaudhry A,Farah MH,et al.Hedgehog signal-ing promotes medulloblastoma survival via Bc/II.Am J Pathol,2007,170(1):347-35518Kenney AM,Rowitch DH.Sonic hedgehog promotes G (1)cyclin expression and sustained cell cycle progression in mammalian neuronal precursors.Mol Cell Biol,,(3)556G L,S f MS,Ty L cells through regulating cell apoptosis and cell c cle.ur studies could provide some clues to develop new therapeutic strateg or .2000202:90-90719artel A er as ner A .p21-negative regulatorof the cell cycle.Proc Soc Exp Biol Med,1996,213(2): 138-14920Yoon JW,Kita Y,Frank DJ,et al.Gene expression pro-filing leads to identification of Gli1-binding elements in target genes and a role for multiple downstream pathways in Gli1-induced cell transformation.J Biol Chem, 2002,277(7):5548-555521Xie J,Aszterbaum M,Zhang X,et al.A role of PDGFRalpha in basal cell carcinoma proliferation.Proc Natl Acad Sci U S A,2001,98(16):9255-925922Teh MT,Wong ST,Neill GW,et al.FOXM1is a down-stream target of Gli1in basal cell carcinomas.Cancer Res, 2002,62(16):4773-478023Ohta M,Tateishi K,Kanai F,et al.p53-independent nega-tive regulation of p21/cyclin-dependent kinase-interacting protein1by the sonic hedgehog-glioma-associated onco-gene1pathway in gastric carcinoma cells.Cancer Res, 2005,65(23):10822-1082924Mao L,Xia YP,Zhou YN,et al.Activation of sonic hedgehog signaling pathway in olfactory neuroblastoma.Oncology,2009,77(3-4):231-24325Morton JP,Mongeau ME,Klimstra DS,et al.Sonic hedgehog acts at multiple stages during pancreatic tu-morigenesis.Proc Natl Acad Sci USA,2007,104(2): 5103-5108(Received May8,2009)。