| BACKGROUNDS AND OBJECTIVES：Osteosarcoma is a malignant tumor which originated frommesenchymal stem cells, accounting for20%of primary malignant bonetumors. About70%-80%patients is diagnosis at the first decade and theannual incidence is about (1-3)/100million. Osteosarcoma usually happensin long metaphysis and associated with poor prognosis. Osteosarcoma iseasy to be confused with trauma because there are almost no clinicalsymptoms, only pain and swelling, sometimes with joint dysfunction, veryfew with pathological fracture treatment.10-20%patients with metastasiswhen diagnose. Before1970, the amputation operation was the majortreatment and five years survival rate had been increased whenchemotherapy was used for osteosarcoma. In the recent30years, peoplewere trying to use new drugs or drug combinations, but the survival rate was not increased. Therefore, it is very important to clarify thepathogenesis and molecular mechanisms of bone sarcoma to improve thesurvival rate.So far, the molecular mechanisms of osteosarcoma have not beenelucidated. And a variety of data suggest that osteosarcoma is a kind ofstem cell differentiation disorders. The incidence of osteosarcoma will beincreased as some genetic mutation or genes deletion.Some genetic diseases, such as retinoblastoma, Li-Fraumeni diseaseoften cause secondary osteosarcoma, and these two diseases are lack of RB,P53gene respectively. Cytogenetic studies on osteosarcoma showedmultiple genetic changes. Abnormal expression of tumor suppressor genesare very important to osteosarcoma.Among them, RB, P53and Kras gene are mostly related withosteosarcoma, but its pathogenesis has not been elucidated. BMP9(Bonemorphogenetic protein, BMP9) is the strongest osteogenic differentiationfactor of mesenchymal stem cells. Ad-BMP9infected iMEF (immortalizedmouse embryonic biochemical fibroblasts cells) cells can experience theosteogenic differentiation. Whether the RB, P53and Kras genes caninfluence iMEF differentiation and result in osteosarcoma is until unknown.The aims of this paper are to investigate the relationship between thesegenes with the embryonic mesenchymal cells osteogenesis andosteosarcomagenesis. CONTENTS(1) To investigate the relationship between bone differentiation defectsand osteosarcomagenesis in RB KO iMEF.(2) To investigate the relationship between bone differentiation defectsand osteosarcomagenesis in Kras overexpression iMEF cells with orwithout RB gene expression.(3) To investigate the relationship between bone differentiation defectsand osteosarcomagenesis in P53KO iMEF cells with or without Kras geneexpression.METHODS1. Establish iMEF RBfl/flcells: MEF RBfl/flcells were got from13.5days pregnancy mice. Immortalized MEF RBfl/fl(iMEF RBfl/fl) were gotafter retrovirus infection.2. Establish iMEF RB-/-cells: In order to knockout RB gene, iMEFRBfl/flcells were infected with AdR-CRE. These cells were identified asfellowing:1. RT-PCR was used to amplify RB gene;2.Whether iMEF RB-/-still have osteogenesis and adipogegesis ability. RT-PCR was used toidentify the levels of iMEF RB-/-cells, and MTT was used to identified theproliferation ability. ALP(alkaline phosphatase) staining, ALP activity andOil Red O staining were used to judge whether iMEF RB-/-still haveosteogenesis and adipogegesis ability.3. Piggybac system was used to subclone plasmids which express Kras gene (pMPB5-Kras) and RB gene (pMPN-RB) respectively.4. Stable cell lines were established which express RB gene and Krasgene: extraction of plasmid pMPN-RB, pMPB5-Kras, then transfectedinto corresponding cells, according to the different plasmid with differentantibiotics: blasticidin S (BSD)(5day) and G418(14day)were used forselecting pMPB5-Kras and pMPN-RB, respectively. iMEF RB-/-26#/RB,iMEF RB-/-54#/RB and iMEF RB-/-pool/RB which over-express RB geneand:iMEF RBfl/fl/Kras,iMEF RB-/-26#/Kras,iMEF RB-/-54#/Kras andiMEF RB-/-pool/Kras which over-express Kras gene wereestablished.RT-PCR was used to identify the RB and Kras gene expression.5. Inducing differentiation:(1) Adenovirus Ad-GFP, Ad-BMP9, Ad-RFP and AdR-CRE wereamplified.(2) Ad-BMP9was used to induce differentiation: After iMEF cellsadherision, Ad-BMP9was added and the infection efficiency should be30%. Medium was changed after six hours. The control group is Ad-GFP.6. Differentiation markers were detected(1)osteogenic ability were detected: iMEF cells were induced byAd-BMP9to identify the osteogenic ability.A.ALP detection: After induced by BMP9for7days, iMEF cells weredectected by ALP activity assay and ALP staining.B. Calcium salt deposition: After14days culture, the differentiation of iMEF cells were detected by Alizarin Red S staining for calcium salt.(2)Adipogenic ability detection: After induced by Ad-BMP9for14days, Oil Red O was used to detect adipogenic ability. The control groupwas infected with Ad-GFP.7. Mice xenograftsA. Cell preparation: iMEF cells were seeded in100mm Dish with10%FBS DMEM medium, which infected by Ad-GFP, Ad-BMP9(infection efficiency is about30%) respectively. After24hours, theinfected cells were digested with trypsin and washed with1-2ml PBS. Thecells were collected and suspended in100μl PBS for subcutaneousimplantation.B. Subcutaneous/intramuscular inoculation: Different sites of4weeksfemale BALB/C mice were injected with100μL cell suspension(1×106cells). The bilateral limb scapular lateral subcutaneous,subcutaneous, bilateral hip muscles of both thighs were injected with cells.C. Mass isolation: One month after inoculation, all mice weresacrificed and the mass were embeded in formalin containing decalcifyingsolution. After2-3days, the embedded masses were paraffining, slicing anddying.8. Pathological and histological diagnosis with HE and Masson’strichrome staining in subcutaneous and muscle mass. RESULTS1. The RB gene knockout iMEF cells were established successfully:The13.5days pregnant RBfl/flmice was sacrificed and the embryo wasused to get primary MEF RBfl/flcells. After retroviral SSR41#immotalization and AdR-CRE infection, we got iMEF RBfl/flcells. RT-PCRwas used to identify the RB expression in monoclonal cells. The resultsshow that the RB gene was successfully knockout in iMEF RB-/-26#, iMEFRB-/-31#, iMEF RB-/-38#, iMEF RB-/-40#,iMEF RB-/-45#,iMEF RB-/-54#andiMEF RB-/-59#; MTT was used to detect the proliferation of iMEFRB-/-clone,results show that,the fourth and fifth days proliferation of cloneiMEF RB-/-26#and iMEF RB-/-54#increased significantly compared withthe control group.2. iMEF cells have osteogenesis and adipogenesis abilityUsing iMEF RB-/-monoclonal which identify by RT-PCR to detect itsosteogenic and adipogenic ability. The iMEF RB-/-monoclonal weredetected by ALP staining, ALP activity assay and Oil red O staining.Compared with the control group, the ALP staining and ALP activity ofclone iMEF RB-/-26#,iMEF RB-/-31#, iMEF RB-/-38#, iMEF RB-/-40#, iMEFRB-/-45#and iMEF RB-/-54#were decreased to a certain extent and Oil redO staining were increased. Among them, the osteogenic of iMEF RB-/-26#,iMEF RB-/-54#decreased obviously, and the adipogenic of iMEF RB-/-26#,iMEF RB-/-54#increased dramatically. The ALP readings of iMEF RB-/-26# and iMEF RB-/-54#are65530±19067,64215±7673respectively. Comparewith control, these two groups decreased93%(p<0.001),90%(p<0.001)respectively. iMEF RB-/-26#, iMEF RB-/-54#cells were inoculatedsubcutaneously into nude mice. Results show that the control group canform bone subcutaneously, while iMEF RB-/-26#can not and iMEF RB-/-54#was decreased. The adipogenesis of both cloning was enhanced. The resultsof HE staining showed that iMEF RBfl/flforms bone like material, while nobone like material was found in iMEF RB-/-26#and iMEF RB-/-54#group.But there were many fat.These results suggested that RB knockout canblock the osteogenic differentiation of iMEF cells, but enhance theadipogenic differentiation. In summary, iMEF cells have the osteogenesisand adipogenesis ability.3. Osteogenic ability was recoved after RB re-expression in iMEFRB-/-cellsPiggybac system was used to re-express RB gene (pMPN-RB). iMEFRB-/-26#and iMEF RB-/-54#were transfected by pMPN-RB and screenedby G418to get stable cell lines. RT-PCR was used to detect the RB level instable cell lines. The cells which re-express RB gene were called iMEFRB-/-26#/RB, iMEF RB-/-54#/RB. We found that in these two clones, ALPstaining, ALP activity reading and Alizarin Red S staining weresignificantly increased. The value of iMEF RB-/-26#/RB ALP activity is9.6times of the control group iMEF RB-/-26#(P<0.001). The value of iMEF RB-/-54#/RB ALP activity is5.5times of the control group iMEFRB-/-54#(P<0.05). Adipogenesis had no change in these two clones.We useiMEF RB-/-26#/RB for in vivo experiment to investigate the osteogenesis innude mice. The results suggested that when RB was re-express in iMEFRB-/-26#, the osteogenic ability was recovered. HE staining results showedthat RB re-expression group format bone like material in the mice muscles.These results showed that osteogenic ability was recoveried when RB genere-expression.To avoid the single cloning difference, all iMEF clones(iMEF RB-/-pool) were pooled and identified as discribe priviously. Theosteogenesis and adipogenesis of iMEF RB-/-pool and iMEF RB-/-pool/RBwere identified. The results showed that the ALP staining, Alizarin red Sstaining and Alizarin activity of iMEF RB-/-pool was significantly lowerthan iMEF RB-/-pool/RB group. Among them, the value of iMEFRB-/-pool/RB was643013±48037. Compare to the control group, the valuewas dreased93%(P<0.001). In vivo experiments are consistent with invitro. HE staining showed that iMEF RB-/-pool/RB format bone likematerial obviously; Masson ’s Trichrome staining showed that iMEFRB-/-pool/RB group formed a large number of red collagen. Bonedifferentiation defect was found in iMEF RB-/-pool group in vivo. Theseresults suggested that when RB re-expression in iMEF RB-/-pool cells, theosteogenic ability were recovered.4. RB KO combined with Kras over-expression can block iMEF osteogenic differentiation and lead to fibrosarcomaiMEF RB-/-26#, iMEF RB-/-54#and iMEF RBfl/flwere transfected withpMPB5-Kras respectively. After BSD selection, the levels of Kras weredetected by RT-PCR in these stable cell lines. The Kras gene overexpression positive cells were named iMEF RB-/-26#/Kras, iMEFRB-/-54#/Kras and iMEF RBfl/fl/Kras. In vitro results showed that the ALPactivity reading and Alizarin Red S staining of iMEF RB-/-26#/Kras, iMEFRB-/-54#/Kras were both decreased compare with control group. Amongthem, the reading of ALP is84590±7100and67661±11836respectively.Compared with the control group, the ALP reading of iMEF RBfl/fl/Kraswas decreased (P>0.05). Compared with the iMEF RB-/-54#/Kras clone, theosteogenic ability in iMEF RB-/-26#/Kras was decreased obviously. AndiMEF RB-/-26#/Kras were used for the next experiment. HE stainingshowed that iMEF RB-/-26#/Kras formed fibrosarcoma and the specimenwas uniform, bundle and arranged in a herring bone like style. Cell volumeis relatively large with rich cytoplasma.A single cloning does not represent the overall characteristics, and wepool iMEF RB-/-cells for Kras over-expression. Results showed that theALP activity, Alizarin red S staining of iMEF RB-/-pool/Kras was decreasedcompare with control group. The ALP activity reading in iMEFRB-/-pool/Kras was decreased93%compared with the control group(p<0.001). HE staining showed that the iMEF RB-/-pool/Kras form fibrosarcoma. HE staining showed that iMEF RB-/-pool#/Kras formedfibrosarcoma and the specimen was uniform, bundle and arranged in aherring bone like style. Cell volume is relatively large with richcytoplasma.In order to knockout RB gene, AdR-CRE was used to infect iMEFRBfl/fl. In vivo results show that AdR-CRE treated iMEF RBfl/flcan formfibrosarcoma; HE staining showed fibrosarcoma formation and Masson ’sTrichrome staining showed that the fibrous tissue was mixed with a paleblue material. These means that RB was not completely deleted afterAdR-CRE infected. Some iMEF cells still have osteogenic ability. The lightblue material means immature bone because RB was not completelydeleted in iMEF RBfl/flcells. HE staining showed that the specimen wasuniform, bundle and arranged in a herring bone like style. Cell volume isrelatively large with rich cytoplasma.5. RB gene combined with Kras block iMEF osteogenic differentiationand form osteosarcoma.In Kras over-expressed and RBfl/fliMEF cells, ALP activity wasdecreased compared with the control group (P>0.05). Alizarin Red Sstaining was deeper than control group which indicates that the iMEFRBfl/fl/Kras has strong osteogenic ability. The combination of HE andMasson’s Trichrome staining results show that iMEF RBfl/fl/Kras in nudemice can form osteosarcoma. 6. P53gene deleted alone or over-express with Kras block iMEFosteogenic differentiation and form fibrosarcoma.For ALP staining, there were less purple materials in iMEF P53-/-andiMEF P53-/-/Kras group. The ALP activity of iMEF P53-/-is about2.1times more than iMEF P53-/-/Kras (P<0.05). For Alizarin Red S staining,there were almost no red materials. These results indicated that iMEF P53-/-and iMEF P53-/-/kras osteoblastic differentiation were blocked even withBMP9stimulation. In vivo results showed that those two groups can formfibrosarcoma in nude mice. HE staining showed that the specimen wasuniform, bundle and arranged in a herring bone like style. Cell volume isrelatively large with rich cytoplasma.Conclusion1.During osteogenic differentiation,RB may be needed for thisprocess.RB gene Knockout only can’t lead iMEF transform toosteosarcoma.2.RB Knockout iMEF with Kras over-express can be transformed tofibrosarcoma;RB nomal iMEF with Kras over-express can be transformedto osteosarcoma.It indicated that:1)During osteosarcoma transfomation,RBmay be needed for this process,2)Duing osteosarcoma transfomationcaused by osteogenic differentiation defect,RB gene can’t lose too early. BACKGROUNDS AND OBJECTIVES：Osteosarcoma is a malignant tumor which originated from bonemesenchymal tissues, accounting for20%of primary malignant bonetumors. About70%-80%patients with onset age at ten to twenty and theannual incidence is about (1-3)/100million. Osteosarcoma usually happensin long metaphysis and associated with poor prognosis. Osteosarcoma iseasy to be confused with trauma because there are no typical clinicalsymptoms, only pain and swelling, sometimes with joint dysfunction, veryfew with pathological fracture treatment.10-20%patients were metastasisat the time of diagnosis. Before1970, the amputation operation was themain treatment methods. Five years survival rate has been increased whenchemotherapy was used for osteosarcoma. In the recent30years, peoplewere trying to use new drugs or drug combinations, but the survival ratewas no longer increased. Therefore, it is very important to clarify thepathogenesis and molecular mechanisms of bone sarcoma to improve the survival rate.The S100family has EF type molecular structure, has21members,can combine with calcium and ion and its target proteins. It plays manyphysiological functions in calcium homeostasis, cell proliferation anddifferentiation, enzyme activity and transcription factor regulation, proteinphosphorylation etc. About15members locate on human unstablechromosome lq21and prone to chromosome rearrangements and geneamplification. It has been found that the abnormal expressions of multiplemembers of the S100are closely related to tumor proliferation, invasion,metastasis and apoptosis. Reports suggest that S100A4also relate with thecancer research reports. THE research found that S100A4expression inhighly invasive MG63.2was higher than that of MG63, suggesting thatS100A4may be in the regulation of the occurrence and development ofosteosarcoma in which plays an important role. In this paper, the purpose ofthe experiment is to investigate the effect of S100A4on the proliferationand osteogenic differentiation regulation role of OS.CELL LINES1. OS cell lines MG63, MG63.2(highly invasive MG63.2wasestablished by our lab),143B2. Mesenchymal stem cells C3H10T1/2.METHODS1. Reverse transcription PCR (reverse transcription PCR, RT-PCR) was used to detect the expression of S100A2, S100A4, S100A6, S100A7,S100B gene in MG63and MG63.2OS cells.2.RT-PCR results indicated that S100A4is the most obvious differentgene between MG63and MG63.2.Adenovirus Ad-S100A4andAdR-siS100A4were established.143B cells were infected byAd-S100A4.S100A4expression was detected by RT-PCR, Westernblot in143B.3. Cell proliferation test: cell doubling time determination was used toanalyze.MG63.2and143B cells were infected with Ad-S100A4,Ad-siS100A4, or Ad-RFP. Cells were replated at72hours in1%FBScomplete media. Cells were then collected by trypsinization at the indicatedtime points and viable cells were stained with Trypan Blue and counted.4. Cell apoptosis test: cell apoptosis was detected by flow cytometry.Subconfluent143B and MG63.2cell were were infected with Ad-S100A4,Ad-siS100A4, or Ad-RFPCells were collected by trypsinization and fixedwith70%ethanol, washed with PBS, and stained with propidium iodideand annexin V-GFP fusion.Cells were subjected to FACS analysis using theBDTM LSR II Flow Cytometer and the FlowJo software.5. Invasion and migration ability by cell scratch test and Transwellassay:Subconfluent143B and MG63.2cell were were infected withAd-S100A4, Ad-siS100A4, or Ad-RFP for16h and replated in6-well cellculture plates. After6h an artificial scratch was made in the center of the cell monolayer using pipette tips. Bright field images of the three differentfields were taken between0h and36h to assess cell migration across thegap.Transwell cell invasion assay:Subconfluent143B and MG63.2cellswere infected with Ad-S100A4, Ad-siS100A4, or Ad-RFP and plated in theupper chambers pre-coated with matrigel. After72h, the membranescontaining the invading cells were fixed in10%formalin, stained withhematoxylin, and mounted onto slides with Permount.6. Detection of osteogenic ability:1)ALP activity was assessed by amodified Great Escape SEAP Chemiluminescence assay and/orhistochemical staining assay (using a mixture of0.1mg/ml napthol AS-MXphosphate and0.6mg/ml Fast Blue BB salt).2) IHC staining:Subconfluentcells were infected with Ad-S100A4, Ad-siS100A4or Ad-RFP. At theendpoint of assays, cells were fixed, permeabilized, and blocked withdonkey serum. Cells were then probed with primary antibodies (caspase-3,OPN and OCN antibodies), followed by incubation with respectivesecondary antibody conjugated with horseradish peroxidase. The protein ofinterested was visualized by3,3’-diaminobenzidine (DAB) stainingRESULTS1. Elevated S100A4expression level in the metastatic MG63.2OSline To test if S100proteins play any role in OS growth and progression, wefirst detected the endogenous expression of S100A2, S100A4, S100A6,S100A7and S100B in human OS line MG63and its metastatic sublineMG63.2using semi-quantitative RT-PCR analysis. The MG63.2line is ahighly metastatic cell line established from its less metastatic parentalMG63line through serial passage in nude mice. Except for S100A7, fourof the examined S100genes were expressed in MG63and/or MG63.2lines.However, only S100A4expression was significantly elevated in MG63.2cells when compared to that in MG63cells, suggesting that high expressionof S100A4may be associated with OS tumor progression and metastasis.2. Silencing S100A4expression inhibits human OS cell proliferationWe next assessed the effect of exogenous S100A4expression orsilencing S100A4expression on OS cell proliferation in143B cell line,which is also a commonly-used and aggressive human OS line. S100A4overexpression increased the proliferation rate of143B cells, with anincrease in the number of cells over that of the RFP control starting day3(p <0.03), while silencing S100A4expression significantly inhibited theproliferation rate of143B cells (p<0.001). A similar increase inproliferation, although to a lesser extent, was seen when S100A4wasoverexpressed in MG63.2cells (p<0.05). However, S100A4knockdownresulted a pronounced decrease in proliferation rate of MG63.2cells(p<0.001). The calculated doubling time (in hours) for S100A4, RFP, and siS100A4-transduced143B cells were38.5±1.1,46.3±2.5, and64.7±3.5,respectively. Accordingly, the doubling time for the MG63.2cellstransduced with RFP, S100A4, and siS100A4were39.9±1.0,37.4±2.0, and45.0±2.3, respectively. Taken together, the proliferation assay resultsindicate that silencing S100A4expression significantly decreases theproliferation rate of OS.3. Silencing S100A4expressin induces OS cells apoptosisWhen143B cells were transduced with AdRFP, AdR-S100A4, orAdR-siS100A4and subjected to FACS analysis with annexin V labeling,the cells were sorted into3phases (viable, early apoptotic, and lateapoptotic). Consistent with the results from the proliferation assays,significantly lower numbers of S100A4-transduced cells were in the earlyand late phases of apoptosis (5.4%and4.5%, respectively) when comparedto the siS100A4group (4.1%and91.5%, respectively, p<0.001).Furthermore, immunohistochemical staining with strong positive stainingof the cleaved caspase-3. The average of cleaved caspase-3positive cellswas significantly higher in S100A4knockdown cells (p<0.001)4.S100A4promotes OS cell migration and invasionThe invasiveness of OS cells was also assessed using a previouslydescribed transwell invasion assay..Subconfluent143B cells were infectedwith Ad-S100A4, Ad-siS100A4, or Ad-RFP and plated in the upperchamber of matrigel-coated transwells for72h. We found that S100A4 overexpression led to a significantly more migrated cells than that of theRFP control group, while silencing S100A4expression in OS cells yieldedthe least invading cells among the three groups. When the numbers ofmigrated cells were quantified, we found that knockdown of S100A4significantly inhibited cell invasion (p <0.001), while overexpression ofS100A4resulted in significantly greater cell invasion compared to the RFPcontrol (p<0.05). Taken together, these results strongly suggest thatS100A4may play an important role in regulating OS cell migration andinvasiveness.6. Silenting S100A4can promote OS, MSC osteogenic differentiationWe and others have demonstrated that osteosarcoma developmentmay be in part caused by differentiation defects, in which mesenchymalstem cells (MSCs) fail to undergo terminal osteogenic differentiation. Thus,we tested the effects of S100A4overexpression and knockdown onosteogenic differentiation of OS cells and mesenchymal stem cells (MSCs).When MG63.2OS cells were transduced with Ad-RFP, Ad-S100A4andAd-siS100A4and early osteogenic marker alkaline phosphatase (ALP)activity was measured at days3and5, we found that knockdown ofS100A4significantly increased ALP activity when compared with RFPcontrol group (p<0.001). We further transduced the MSC line C3H10T1/2cells with Ad-RFP, Ad-S100A4, and AdR-siS100A4, and found thatS100A4knockdown induced a significant increase in ALP activity at day7. In both OS and MSC cells, S100A4overexpression did not induce anysignificant changes in ALP activity when compared with that of the RFPcontrol. Lastly, we examined the effects of S100A4expression on the lateosteogenic markers osteopontin (OPN) and osteocalcin (OCN). Consistentwith the results obtained in ALP assays, knockdown of S100A4expressionin MG63.2cells resulted in a significant increase in the expression of OPNand OCN, while S100A4overexpression and RFP expression resulted inminimal staining. These results suggest that S100A4may play an importantrole in regulating the intricate balance between proliferation anddifferentiation of osteoblast progression.CONCULUSIONOur results strongly suggest that downregulate S100A4may inhibitOS tumor growth by regulating the cell cycle, reducing apoptosis, andpromoting osteogenic differentiation. Thus, S100A4may serve as a markerfor tumorigenic potential, as well as a therapeutic target... |
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