P53-induced Gene Unc5H4 In The Tumorigenesis Of Neuroblastoma

IntroductionNeuroblastoma(NB)is common solid tumor in childhood.The mortality of NB is very high,even recently with the intensive chemotherapy,radiotherapy and bone marrow transplantation,the survival rate of malignant NB is still less than 10%.Typeâ… transmembrane receptors such as DCC(Deleted in Colorectal Cancer)and UNC5H have been considered to belong to the so-called dependence receptor family. These receptors share functional similarity to promote apoptosis without their respective ligands including netrin family,but inhibit apoptosis when bound to these ligands.As described,DCC was one of caspase substrates and served as a caspase amplifier under conditions in which the ligand is unavailable.Extensive studies suggested that DCC acts as a tumor suppressor,however,DCC is rarely mutated in human cancers.Mammalian UNC5H family is composed of UNC5H1-4 Among them,UNC5H4 has been recently found in human genome database Thiebault et al.described that expression levels of UNCSH1-3 are strongly down-regulated in various primary tumors which is associated with loss of heterozygosity(LOH)within UNCSH loci,and enforced expression of UNC5H1, UNC5H2 or UNC5H3 inhibits malignant transformation,which is related to their pro-apoptotic activity.According to their results,UNC5H-mediated apoptosis was dependent on their cytoplasmic death domain,and potent caspase inhibitor abrogated their pro-apoptotic activity.Consistent with these observations,UNC5H1-3 contained classic caspase cleavage site(DXXD)and caspase-mediated cleavage of UNC5H was required for cell death induction.Although UNC5H family has pro-apoptotic activity,the precise molecular mechanisms behind UNC5H-mediated apoptosis remained unclear.Of note,UNC5H2 is a direct transcriptional target of p53 and UNC5H2-mediated apoptosis is regulated in a p53-dependent manner.Based on their results,netirin-1 inhibited p53-dependent apoptosis without affecting expression levels of p53. Alternatively,Llambi et al.found that UNC5H2 interacts with death-associated protein kinase(DAP-kinase)and their interaction enhances catalytic activity of DAP-kinase. Intriguingly,DAP-kinase required functional p53 for induction of apoptosis.In contrast to UNC5H1-3,little is known about functional significance of UNC5H4.In this study,we found that UNC5H4 is a direct transcriptional target of p53,and UNCSH4-mediated apoptosis is dependent on p53 status.Methods一,Clinical Section1.Clinical Materials:We first studied 56 cDNA samples of primary2.Neuroblastoma including 16 favorable Neuroblastma and 16 unfavorable Neuroblastoma for the expression patterns of Unc5H1-4.The ages of those patients were from 1 month to 14-year old,with average 3.1 years old.Those samples came from Chiba Research Institute of Japan,which is the sample bank of Neuroblastoma in Japan and the usage of those samples and clinical data were fully matched with the relative Japanese laws.3.RT-PCR:RT-PCRwas performed to detect the expression of Unc5H1,2,3,44.in those 32 Neuroblastoma cDNA to clarify the expression patterns for those genes in Neuroblastoma.5.Real-time PCR:118 cDNA of primary Neuroblastoma were also provided6.from Chiba Cancer Research Institute as well as the clinical data.Real-time PCR were done for the expression of UncSH1,3,4.7.Kaplan-Meier survival analysis:The expression of Unc5H1,3,4 and8.also analyse with the clinical data,Kaplan-Meier survival curve was made to show the connection of gene expression and the clinical outcome. 二,Basal Section1.Cell lines and culture:Human osteosarcoma-derived U2OS and SAOS-2 cells were maintained in Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%heat-inactivated fetal bovine serum,penicillin(50 U/ml)and streptomycin(50μg/ml).Human lung carcinoma-derived H1299 cells were cultured in RPMI 1640 medium supplemented with 10%heat-inactivated FBS and antibiotic mixture.Cells were grown at 37℃in a humidified atmosphere of 5%CO₂ in the air.Where indicated, cells were exposed to adriamycin(ADR)at a final concentration of 1μM.2.Transfection:Cells were transfected with the indicated expression plasmids using LipofectAMINE 2000 transfection reagent(Invitrogen)according to the manufacturer's instructions.3.Cell survival assay:Cells were seeded at a density of 5×10³ cells/96-well cell culture plates and allowed to attach overnight.Cells were then treated with 1μM of ADR.At the indicated time points after ADR treatment,10μl of a modified 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide solution(Dojindo)were added to the culture and reaction mixtures were incubated at 37℃for 1h.The absorbance readings for each well were carried out at 570 nm using the microplate reader(Model 450,Bio-Rad Laboratories).4.RT-PCR:Total RNA was isolated from the indicated cells by using RNeasy Mini Kit(Qiagen)according to the manufacturer's recommendations and reverse transcribed in the presence of random primers and SuperScriptâ…¡reverse transcriptase (Invitrogen).The resultant first-strand cDNA was amplified by PCR to measure the expression levels of genes of interest.PCR products were separated by 1.5%agarose gel electrophoresis and visualized by ethidium bromide staining.5.Immunoblotting:Cells were washed in ice-cold phosphate-buffered saline (PBS)and lysed in SDS-sample buffer containing 10%glycerol,5%-mercaptoethanol, 2.3%SDS and 62.5 mM Tris-HCl(pH 6.8).The protein concentration of cell lysates was determined by using Bio-Rad protein assay dye reagent(Bio-Rad Laboratories) according to the manufacturer's instructions.Bovine serum albumin(BSA)was used as a standard.Equal amounts of cell lysates were separated by 10%SDS-polyacrylamide gel electrophoresis,electro-transferred onto Immobilon-P membrane filters(Millipore) and blocked with 0.3%non-fat milk in Tris-buffered saline(TBS)containing 0.1% Tween 20 at 4℃.The membranes were probed with first antibody at room temperature for 1 h followed by incubation with horseradish peroxidase-conjugated secondary antibodies(Cell Signaling Technology)at room temperature for 1 h.Immunoreactive bands were visualized by using ECL system(Amersham Biosciences)according to the manufacturer's instructions.6.Establishment of p53-knoced down cell clones:U2OS cells were transfected with the empty plasmid(pSUPER,OligoEngine)or with the expression plasmid for siRNA against p53(pSUPER-p53).Forty-eight hours after transfection,cells were transferred into the fresh medium containing G418(Sigma)at a final concentration of 500μg/ml and incubated for 2 weeks.Then,G418-resistant clones were picked up and cultured in the presence of G418.Expression levels of p53 in each cell clone were analyzed by immunoblotting.7.Construction of luciferase reporter plasmids:The indicated luciferase reporter constructs driven by putative p53-responsive elements of UNC5H4 gene.The resultant PCR products were gel-purified and inserted into the appropriate restriction sites of pGL3-promoter plasmid(Promega)to give p53-RE1,p53-RE2,p53-RE3 and p53-RE4. The constructs were verified by DNA sequencing(Applied Biosystems,).8.Luciferase reporter assay:p53-deficient H1299 cells were seeded at a density of 5×10⁴ cells/12-well cell culture plates and allowed to attach overnight.Cells were transiently co-transfected with 100 ng of pGL3-Promoter plasmid(Promega),p53-RE1, p53-RE2,p53-RE3 or p53-RE4,10 ng of Renilla luciferase reporter construct(pRL-TK, Promega)and 25 ng of the expression plasmid for FLAG-p53.Total amount of plasmid DNA per transfection was kept constant(510ng)with pcDNA3.Forty-eight hours after transfection,cells were lysed and their luciferase activities were measured by using Dual-Luciferase Assay System(Promega)according to the manufacturer's instructions. The firefly luminescence signal was normalized based on the Renilla luminescence signal.9.Chromatin immunoprecipitation(ChIP)assay:ChIP assay was performed as described[16].In brief,H1299 cells were transfected with the empty plasmid or with the expression plasmid for p53.Forty-eight hours after transfection,cells were cross-linked with 1%formaldehyde in medium for 10 min at 37℃.Cross-linked chromatin was prepared from cells and sonicated to an average length of 200-800 nucleotides, precleaned with salmon sperm DNA/protein A-agarose beads,and immunoprecipitated with normal mouse serum(NMS)or with monoclonal anti-p53 antibody.The immunoprecipitates were eluted with 100 1 of elution buffer(1%SDS and 1 mM NaHCO₃).Formaldehyde-mediated cross-links were reversed by heating at 65℃for 4 h,and the reaction mixtures were treated with proteinase K at 45℃for 1 h.Genomic DNA was purified using the QIAquick PCR purification kit(Qiagen).Purified DNA was amplified by PCR.10.Colony formation assay:U2OS and H1299 cells were transfected with the empty plasmid(pcDNA3)or with the expression plasmid encoding UNC5H4. Forty-eight hours after transfection,cells were transferred into the fresh medium supplemented with G418(400μg/ml).After two weeks of selection,drug-resistant colonies were stained with Giemsa's solution,and number of drug-resistant colonies was scored.11.Flow cytometry:Forty-eight hours after the treatment with ADR(at a final concentration of 1μM),floating and attached cells were collected,washed in ice-cold PBS and fixed in 70%ethanol at -20℃.The cells were washed in ice-cold PBS and resuspended in phosphate-citrate buffer(4 mM citric acid and 200 mM Na₂HPO₄)and kept at room temperature for 15 min.Nuclear DNA was stained with propidium iodide (40μg/ml)in the presence of RNase A(10μg/ml)and the reaction mixture was incubated in the dark for 30 min.After the incubation with propidium iodide,DNA content of cells was examined by FACScan flow cytometer(Beckton Dickinson)using CellQuest software.Results一,Clinical Section1.Unc5H4 is highly expressed in favorable NB:With RT-PCR,in 16 favorable NB cDNA and 16 unfavorable NB cDNA,it was found that Unc5H1,3,4 seemed to express higher in favorable NBLs.Using 118 cases of cDNA from primary NBLs,we found only Unc5h4 was highly expressed in favorable NBLs,while Unc5H1 and Unc5H3 were no significance.2.In all Neuroblastoma the higher Unc5H4 expressed the longer survival:3.In unfavorable or MYCN amplification NB,the higher Unc5H4 expressed,the longer survival time.二,Basal Section1.Unc5H4 inducescell death including Neuroblastoma cells.2.The induction of apoptosis by Unc5H4 depends on normal p53 status.3.There are p53 binding sites in intron 1 region of Unc5H4.Results1.Unc5H4 has higher expression in favorable Neuroblastoma than that in unfavorable ones.2.Higher expression of Unc5H4 relates with longer survival rate.3.Unc5H4is a newly identified director of Neuroblastoma.4.Unc5H4 can induce apoptosis of Neuroblastoma cells and this function depends on normal p53 status.5.Unc5H4 is a direct transcriptional target of p53.