Experimental Studies Of Therapeutic Effect Of CD::UPRT/5-FC Unification Bi-gene Therapy On Glioma By Retrovirus-mediated

The purpose is to study anticancer effects of fusion suicide geneCD::UPRT/5-FC on glioma and the pharmacokinetic of 5-FC in thesystem of CD::UPRT/5-FC gene therapyPart 1: Construction of the recombinant retrovirus vector expressingCD/UPRT and harvest the recombinant retrovirus with CD::UPRTgene.Objective: To construct and harvest the recombinant retrovirus withCD::UPRT gene.Methods: Two pairs of PCR primers for CD::UPRT genes were designedaccording to the multiclone sites of retroviral expression vectors, pLXSNand LZRSpBMN-Z, and the sequence of CD::UPRT. EcoR I and BamHI sites were interpolated at the 5 ends of the primers respectively.CD::UPRT genes were amplified from the plasmids, pORF5-CD::UPRT,by PCR. After their DNA sequencing analysis, CD::UPRT genes were further inserted into the sense open reading frames of pLXSN andLZRSpBMN-Z. The CD::UPRT inserted in recombinant expressionplasmids were confirmed by enzymes digestion and electrophoresis. Thenthese plasmids transfected packing cells, PT67 and Pheonix. Supernatantof the cloned packing cells were harvested and detected their titers of therecombinant retroviruses.Results: It was confirmed by enzyme digestion and electrophoresis thatCD::UPRT genes were inserted recombinant retrovirus plasmids(pLXSN-CD::UPRT, LZRS-CD::UPRT). The sequence of CD::UPRT is1227bp, identical to the gene order of protogene. The virus titer of theharvested retrovirus were as following: LZRS-GFP, 2×10⁶CFU/ml;LZRS-CD::UPRT, 2×10⁵CFU/ml; pLXSN, 3×10⁶CFU/ml;pLXSN-CD::UPRT, 1×10⁵CFU/ml.Conclusion: The recombinant retrovirus expressing CD::UPRT(LZRS-CD::UPRT, pLXSN-CD::UPRT) were constructed, and theretrovirus with CD::UPRT were harvested with high titers.Part 2: Establishment and identity of the gene recombinant C6 celllines with stable expression of CD/UPRTObjective: To establish the new gene recombinant C6 cell linesexpressing CD/UPRT stably for CD::UPRT/5-FC gene therapy study. Methods: For 30 minutes at 32℃1000g centrifugalaization, C6 Cellswere infected by the recombinant retroviruses with CD::UPRT andpLXSN that were constructed in Partâ… . The cells were selected byneomycin (G418) at the final concentration 200ug/ml. During the process,hexadime bromide was added to 8ug/ml concentration into the infectingsystem to assist the recombinant retrovirus infective rate. After 6 hrs, theculture fluid was refreshed. Another 24h hrs culture was going and theinfecting process was repeated ad un vic. The cells were cultured for 48hrs and then G418 was added into culture fluid to the final concentration200ug/ml. The cell clones of anti-G418 were selected under G418 for 14days. These C6 cell lines containing CD::UPRT or pLXSN genes werenamed C6-CD::UPRT and C6-pLXSN respectively. Introduced andinserted genes were confirmed by electrophoresis. The biologicalcharacteristics of C6-CD::UPRT were compared with the ones ofpro-generation C6.Results: By respective cDNA templates of C6-CD::UPRT, C6-pLXSN,and PCR and RT-PCR with the two sets of primers for those cell lines,1ï¼…agarose gel electrophoresis demonstrated respectively that CD::UPRTgenes had been integrated into the chromosome of resistant cells,C6-CD::UPRT. Specific bands, CD::UPRT andβ-actin were shown at1.2Kb and 661bp for C6-CD::UPRT cells. For C6-pLXSN, only one band,β-actin was found at 661bp. There were no obvious differences in biological characteristics between transferred genes cells, C6-CD::UPRTand their pro-generation cells, C6.Conclusion: The cell lines, C6-CD::UPRT were established, which werewith the same biological characteristics as C6, but they can expressCD::UPRT stably.Part 3: The study of the therapeutic effect and mechanism ofCD::UPRT/5-FC gene therapy system on glioma cells in vitroObjective: To study the therapeutic effect and mechanism ofCD::UPRT/5-FC gene therapy system on C6 glioma cells in vitro.Methods: The cells of C6-CD::UPRT, C6-pLXSN and C6 were culturedrespectively and then 5-FC was administered into their culture fluids. Thechanges of the cells of were respectively observed through electronmicroscopy. The 5-FC cytotoxicity efficacies to these cells wereevaluated by MTT methods. Then the growth inhibition rates (GIR) werecounted. Immunohistochemical assay (SABC method) was used to detectthe expression of Fas,FasL and Bcl-2, and the apoptosis was analyzed byflow cytometry for these cultured cell lines.Results: The fusion bi-suicide-gene, CD::UPRT combined with5-fluorocytosine could significantly kill C6-CD::UPRT. Its GIR wasremarkable increase comparing with C6 and C6-pLXSN cells at the final concentration of 5-FC≥10ug/ml (p＜0.001). The growth inhibition rates ofC6-CD::UPRT cells were 30ï¼…, 63ï¼…and 75ï¼…at the final concentrationsof 10ug/ml, 100ug/ml, and 1000ug/ml 5-FC respectively. However, thegrowth inhibition rates of C6 and C6-pLXSN cells were no obviousdifference at these different 5-FC concentrations. Undergoing 5-FCadministration, the percentages of apoptosis in C6-CD::UPRT cells weremuch higher than that of the other two cell lines according to calculationby the flow cytometry (p＜0.005). The electron microscope examinationshowed that there were apoptotic bodies in gene-transferred cells,C6-CD::UPRT. That suggested that apoptosis might play an importantrole in killing mechanism. Immunohistochemical assay demonstrated thedown regulation of Fas, Fas-L and Bcl-2 gene expressions after theadministration of 5-FC in C6-CD::UPRT cells. All of these suggested thatthe killing mechanism of CD::UPRT/5-FC gene therapy is associatedwith apoptosis, which could be induced by Fas, Fas-L and Bcl-2.Conclusion: The fusion bi-suicide-gene therapy system, CD::UPRT/5-FCcould significantly kill C6-CD::UPRT cells. The killing mechanism ofCD::UPRT/5-FC gene therapy was associated with apoptosis, in whichFas, Fas-L and Bcl-2 might play a role.Part 4: The therapeutic effect of the CD::UPRT/5-FC on glioma innude mice in vivo Objective: To study the therapeutic effect of CD::UPRT/5-FC on thesubcutaneous xenografted glioma in nude mice in vivoMethods: Nude mice were xenografted by glioma cells, C6, C6-pLXSNand C6-CD::UPRT. After the cells were implanted subcutaneously for 7days and the xenografted tumors free grew up a little bit, 5-FC or PBSwas injected intraperitoneally once a day for 10 days continuously. Thetumors' volumes were measured routinely and survival days of the nudemice counted in following days. Finally, the killing efficacies of 5-FC onxenografted tumors in the nude mice were investigated byelectron-microscope and FCM assays.Results: After 5-FC administration, the growth of C6-CD::UPRTxenografted tumor was significantly suppressed, the tumors' volumeswere much smaller and the life spans of C6-CD::UPRT xenograftedtumor nude mice were prolonged distinctly comparing with C6 andC6-pLXSN control groups (p＜0.05). In C6-CD::UPRT group, GIR wasremarkable increase showed by MTT assays and the percentage ofapoptosis calculated by the flow cytometry was much higher than that ofthe controls (p＜0.05). Electron microscopy showed obvious apoptosisbodies in tumor cells of C6-CD::UPRT group. All of the above indicatedthe killing mechanism of CD::UPRT/5-FC gene therapy was associatedwith apoptosis.Conclusion: The CD::UPRT/5-FC gene therapy system can significantly inhibit the growth of subcutaneous xenografted tumors in nude mice invivo. The results showed apoptosis took part in the killing mechanism ofCD::UPRT/5-FC gene therapy.Part 5: Experimental studies of therapeutic effect of CD::UPRT/5-FCunification bi-gene therapy on intracranial xenografted encephalicglioma in rats in vivoObjective: To investigate the therapeutic effects of CD::UPRT/5-FCcombinative gene therapy on intracranial xenografted encephalic gliomain SD rats in vivo.Methods: Reconstructed malignant glioma cells C6-CD::UPRT, or C6were xenografted into the rat's brain by Horseley-Clarke technique understereotaxic apparatus. When tumors grew up for 7 days, the rats weretreated with 5-FC (500mg.kg^-1.d^-1) or PBS intraperitoneally once a dayfor 10 days continuously. The treatment was repeated once after 20 daysof the first 5-FC treatment. The therapeutic effects were determined bymeasuring tumor size, the survival period, and the tumor tissues wereinvestigated by electron microscope and FCM assays.Results: Comparing with control groups (C6 group), the growth of tumorin test group (C6-CD::UPRT group) was significantly suppressed. The average survival periods of the control groups were about 32 days, but thesurvival periods of the test group extended to 85 days (p＜0.01). In the testgroup there were significant apoptotic peaks showed by FCM assays.Comparing with C6 group, apoptotic rate was 19.36ï¼…(p＜0.05).Additionally, the karyon break and apoptotic bodies were widespread inthe cells of the group shown by electron microscope.Conclusion: CD::UPRT/5-FC combinative bi-gene therapy has asignificant therapeutic efficacy on intracranial xenografted encephalicglioma in SD rats in vivo. There were obvious apoptosis phenomenons inthe cells of treated tumors.Part 6: Study of pharmacokinetics and bystander effect and itsmechanism in CD::UPRT/5-FC bi-gene therapy in vitro and in vivoObjective: To investigate pharmacokinetics and to establish anoninvasive, dynamic quantitative method to evaluate the therapy effectin CD::UPRT/5-FC bi-gene therapy strategy in vitro and in vivo. Todetect bystander effect and its mechanismMethods: The pharmacokinetics of CD::UPRT/5-FC bi-gene therapystrategy were investigated by ¹⁹F-MRS analysis to demonstrate the CDand UPRT enzymes expressed by C6-CD::UPRT cells and these enzymeseffect on the transformative process of 5-FC to 5-FU and F-Nuctd. C6 and C6-CD::UPRT cell lines were cultured by RPMI-1640 containing5-FC(1000μmol/L) at 37℃and 5ï¼…CO₂ for 24 hrs. And then themedium, the cells and their commixture were collected separately for¹⁹F-MRS analysis. For ¹⁹F-MRS study in vivo, the rats with intracranialxenograghted encephalic glioma were anesthetized and injectedintraperitoneally with 5-FC, one dose 500mg/kg body weight followed byserial ¹⁹F-MRS spectra studies.Mixtur cells of C6 and C6-CD::UPRT at different ratio were culturedby RPMI-1640 with the final concentration of 1000u mol/L 5-FC. After 4day culture, the cytotoxicity efficacy was evaluated by MTT methods, thesurvival rate of cells was counted, and bystander effect and its mechanismwere studied.Results: After 24hr culture of C6-CD::UPRT cells with 1000μmol/L5-FC, ¹⁹F-MRS spectra of the cells showed three broad resonance wavesat -49.2ppm, -50.6 ppm and -45.5 ppm corresponding to 5-FC, 5-FU andF-Nuctd. In each sample, there were no 5-FU and F-Nuctd at verybeginning. These were produced by CD enzyme catalyzing 5-FC and byUPRT enzyme catalyzing 5-FU. In vivo serial ¹⁹F MRS spectra showedthe fortis 5-FC peaked wave, weak 5-FU one at 20 minute after 5-FCinjection. And then the 5-FU concentration reached a maximum at about50 minute and both became descent within 4-5 hrs. At over 157 minute,these two only were detected as trace signals. The F-Nuctd signal first appeared in about 1 hr, increased steadily and remained detectable 6-24hr after 5-FC injection.With the increase of the ratio of C6-CD::UPRT cells, the survivalrate of mixture cells were decreasing. At the ratio of 10ï¼…, the survivalrate was 79.55±0.88ï¼…with a significant difference. Accompanying withthe C6-CD::UPRT ratio increasing, the survival rate was descent.Conclusion: In vitro and in vivo ¹⁹F MRS results indicated thatC6-CD::UPRT cells can effectively express CD and UPRT enzymes.These two enzymes can effectively directly catalyze5-FC→5-FU→F-Nuctd, which can directly kill the tumor cells. Theobvious bystander effect of the CD::UPRT/5-FC system and 5-FC,5-FU,F-Nuctd appearance in cell culture fluid indicated that active metabolitesof 5-FC could diffuse efficiently from intracellular compartment toextracellular one. And this study demonstrates the ability of ¹⁹F MRS tononinvasively, dynamically and quantitatively estimate the level oftransferred CD::UPRT gene expression and their efficiency of catalyzingsubstrates.