Tumor-specific TK Gene Therapy Induced By HTERT Promoter For CD133+ Human Nasopharyngeal Carcinoma Cells

BackgroundNasopharyngeal carcinoma which has high incidence in southern China and Southeast Asia is one of the serious malignant tumors,it has threated and impacted of people's health and quality of life.At present,the clinical radiation therapy is still the preferred method of the treatment of nasopharyngeal carcinoma,although in recent years,radiation therapy equipment,technologies and methods are at constant progress, the 5-year survival rate of nasopharyngeal cancer and the cure rate remained at 50%,which did not fundamentally improve the treatment effect and radiotherapy in itself brought about by local and systemic side effects also give patients a great physical and psychological harm,which seriously impacted on treatment outcome. Therefore,it is very important to explore the most fundamental cause of nasopharyngeal carcinoma and new effective treatment method to it.In recent years,a new theory of tumor origin,that is,tumor stem cell theory is gradually being recognized by everyone.Cancer stem cells are a small part of cell groups with self-renewal capacity and non-directional ifferentiation potential,which have the nature of stem cells and are present in tumor tissue,it is the fundamental cause of the formation of different degree of tumor cells and tumor growth,tumor stem cell is starting cell in the process of tumorigenesis,extension and recurrence.So far,it has successfully isolated tumor stem cells from leukemia,breast cancer,brain tumors,lung cancer,prostate cancer and other tumor. Since Toru Kondo has been found that tumor stem cells persist in many cancer cell lines,There are certain progress in the study of tumor stem cells about tumor cell line,which is seen as classic model for studying cancer.Zhu found that spheres could isolated from glima cell line under serum-free culture condition containing growth factor,those spheres were enrich in cancer stem cells,so he called them brain tumor spheres.Study abroad has shown that tumor stem cells with high telomerase activity, tumor stem cells and telomerase system have close relationship which may explain the unlimited proliferative potential and potential for malignant transformation of tumor stem cells,namely,high telomerase activity may be the "power source"of supporting the malignant transformation of tumor stem cells,which provides an entry point for our in-depth study.As so far,the report about cancer stem cells in nasopharyngeal carcinoma cell lines is rare.Therefore we choose a human nasopharyngeal carcinoma cell line SUNE as research objective and tried to isolate nasopharyngeal carcinoma stem cells with specifical surface markers CD133 by immunomagnetic bead sorting method,and cultivated nasopharyngeal carcinoma stem cells under special serum-free medium. We identify the unique self-renewal and multidirectional differentiation abilities of tumor stem cells,while detecting its telomerase activity,finally we transfect human telomerase reverse transcriptase promoter/TK specific tumor-specific expressing system(hTERT/TK/pGL3) that we had constructed into nasopharyngeal carcinoma stem cells,which had been isolated by immunomagnetic bead sorting and confirmed,and explore the effect that after the above specifical expression system killed nasopharyngeal carcinoma stem cells,as well as changes in the activity of telomerase system.ObjectiveTo research the expression of CD133 in human nasopharyngeal carcinoma cell line SUNE and detect proliferation and differentiation capabilities in vitro of CD133~+ tumor cells,and identify the nature of tumor stem cells to it;to explore the effect that after the human telomerase reverse transcriptase promoter/TK specific tumor-specific expressing system(hTERT/TK/pGL3) killed nasopharyngeal carcinoma stem cells in nasopharyngeal carcinoma cell line SUNE,as well as changes in the activity of telomerase system.Methods1.Culture of SUNE Cell Line:A nasopharyngeal carcinoma cell line SUNE was cultured in RPMI1640,supplemented with 10%newborn bovine serum in an incubator of 5%CO2 with a temperature of 37℃.daily for liquid,Near confluence (about 80 percent),the cells were trypsinized by 1:2 or 1:3 and rinsed in media PBS. when the cell growth to cultivate the bottom 80 is covered when cultured.2.Detection of Expression of CD133 in SUNE Cell Line by Flow Cytometry: Counting about 10~7 SUNE cells,SUNE cells were trypsinized by 0.25%trypsin and rinsed in 0.01%PBS,the cells were centrifuged at 1000g for 5 minutes.remove supernatant,adding 10μL anti-CD133 labeled with phycoerythrin(phycoerythrim, PE),20μL FcR blocker,80μL buffer,fully fixed,the reactant was incubated in the dark for 10 minutes at the temperature of 4℃,buffer washing,re-suspended, centrifuged and removing supematant,add 500μL buffer to cells,cells were analyzed on Flow Cytometry.3.Immunocytochemical Staining of CD133~+ Tumor Stem Cells:Counting about 5X10~4 SUNE cells,cells were plated onto glass coverslips in RPMI 1640 for 2 to 3 days,when the cell adhered to the dish,the culture medium was added,the cells were fixed with 4%paraformaldehyde for 15 to 20 minutes at room temperature and washed with 0.01%phophate-buffered saline(PBS),blocked by 1%fetal calf serum for 30 minutes.Add 2.5μL anti-CD133 labeled with PE,the reactant was incubated in the dark for 30 minutes at the temperature of 4℃.After this,cells were washed with 0.01%PBS,finally,the glass coverslips was mounted with the buffer glycerol mount and observed under fluorescence microscopy.4.Magnetic Cell Sorting and Flow Cytometry:Counting about 10~7 SUNE cells, cells were trypsinized by 0.25%trypsin and rinsed in 0.01%PBS,and were ccntrifuged at 1000g for 5 minutes.remove supernatant.Cells were then suspended in 300μL of buffer.Adding 100μL FcR blocking reagent,100μL microliters of CD133 MicroBeads to the cells,One hundred microliters of CD133 MicroBeads (Skedsmokorset,Norway) were added to label the cells,fully fixed,the reactant was incubated in the dark for 30 minutes at the temperature of 4℃,cells were washed by adding a buffer and centrifuging at 300 g for 10 minutes,Supernatant was pipetted off,Cell pellets were suspended again in a 500μL buffer.An LS column was placed in the magnetic field of the MACS separator.The column was rinsed with 3 mL of buffer.Cell suspension with 0.5 mL of buffer was applied onto the column,which was then washed with 0.5 mL of buffer three times.The column was then removed from the separator and placed on a collection tube.One milliliter of buffer was pipetted onto the column.The fraction with magnetically labeled cells was firmly flushed out using the plunger supplied with the column.The separation step using MS columns was repeated.CD133~+ sorted cell populations were cultured in two different medium.5.Cell Proliferation Assays In Vitro:The separated CD133~+ tumor cells,CD133~-tumor cells,and unsorted tumor cells were plated in 96-well microwell plates in 0.2 mL of RPMI 1640(supplemented with growth factors) at a density of 2,000 cells/well (Medium without cells was added as a control).Cells were fed with medium every 3 days.Proliferation assays were performed on days 1,3,5,and 7(postplating) using Roche 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.Quantification of viable cells through reading of ultraviolet absorption spectrums at 490 nm was performed on a Versamax microplate reader.The number of days in culture and average data of different groups were used to draw growth curves to compare the proliferation abilities of the three groups.6.Cell Differentiation Assays In Vitro:After isolation,CD133~+ tumor cells, CD133~- tumor cells,and unsorted tumor cells were cultured in RPMI 1640 supplemented with 10%FCS in an incubator of 5%CO2 with a temperature of 37℃. Every group was plated in six microwells.The medium was changed every 2 days.To observe differentiation ability,the percentage of CD133~+ tumor cells was detected using flow cytometry on days 1,3,6,9 and 12.Average data from different groups were used to draw curves of differentiation. 7.Detect telomerase activity of SUNE,CD133~+,CD133~- and ECV cells:First, select four group cells,while counting about 2X106 cells every group,then perform as TRAP-PCR silver staining of telomerase activity detection kit,and finally determine their telomerase activity by comparing with the gray scale of their ladder strap on gel electrophoresis.8.Transiently transfect recombinant plasmid CMV/TK/pGL3,hTERTp/TK /pGL3 and TK/pGL3(our experimental group has constructed and identified) to CD133~+ nasopharyngeal carcinoma stem cells in SUNE cell line with liposomes,then observe their changes of telomerase activity:nasopharyngeal cancer stem cells were plated in 6-well microwell plates at a density of 1X106 cells/well,transfect recombinant plasmid CMV/TK/pGL3,hTERTp/TK/pGL3 and TK/pGL3 to CD133~+ nasopharyngeal carcinoma stem cells in SUNE cell line with liposomes,4-6 hours after transfection add drug GCV,observing for 48 hours,then perform as TRAP-PCR silver staining of telomerase activity detection kit.9.Transiently transfect recombinant plasmid CMV/TK/pGL3,hTERTp/TK/ pGL3 and TK/pGL3 to CD133~+ nasopharyngeal carcinoma stem cells in SUNE cell line and ECV cell with liposomes,then detecting their activity by MTT assay:human nasopharyngeal carcinoma stem cells and the umbilical vein endothelial cells ECV grow to 80%,cells were plated in 96-well microweU plates in 100ul of RPMI 1640 at a density of 1×10~5 cells/well;transfect recombinant plasmid CMV/TK/pGL3,hTERTp /TK/pGL3 and TK/pGL3 to CD133~+ nasopharyngeal carcinoma stem cells in SUNE cell line and ECV cell with liposomes,4-6 hours after transfection add drug GCV,after 48 hours adding MTT,incubated 4 hours,and finally add DMSO.Results1.Cultured in RPMI 1640 supplemented with serum,SUNE cells adhered to the dish and had active proliferation ability.Under a phase-contrast microscope,cells had a fusiform shape,many prominences with large nuclei and ze good brightness.2.Detection of expression of CD133 in SUNE cell line by Flow Cytometry,only 0.35%of SUNE cells expressed the membrane antigen CD133. 3.At cell SUNE glass coverslips,only a few cells showed positive expression of CD133.Under a fluorescence microscope,a glass slide full of cells had sparse light orange-red fluorescence.The fluorescence was globular and stronger in circumference, paralleling the cellular membrane.It could be inferred that CD133 was only expressed in a few cells and CD133 was expressed in the cellular membrane.4.After isolation,CD133~- cells were immediately cultured in serumfree RPMI 1640 medium.Cells grown in these conditions were single,spherical,floating.As days passed,nonadherent single cells became clusters.The cell population also increased,five days after culture,the size of cell clusters.At the same time, suspension cells were centrifuged and cultured in RPMI 1640 medium with 10%FBS. After 24 hours,cells adhered to the dish again.5.After isolation,CD133~+ cells,CD133~- cells,and control unsorted SUNE cells were separately cultured in serum-free medium,and add growth factors to stimulate cell proliferation,On days 1 to 3 and 5 to 7,observe and compare of proliferation activities of three cells,compared with CD133~- cells and control unsorted SUNE cells,CD133~+ cells demonstrated increased proliferation capacity in vitro,especially at 1～3 d and 5～7 d.6.After isolation,single CD133~+ cells were cultured in RPMI 1640 medium supplemented with 10%FBS.immunofluorescence chemical methods can be clearly observed,with the extension of culture time,cell surface antigen CD133 at a gradual reduction orange-red fluorescence that is at the gradual disappearance.At the same time,Consistent with the results of the dynamic flow cytometry detect the proportion of CD133 cells in line with the outcome of immunocytochemistry mentioned above, on days 12,CD133~+ cells from the beginning of the proportion of 89.33±0.882% down to 0.33±0.088%.From experimental results,we can understand that the CD133~+ nasopharyngeal carcinoma cells in vitro differentiation.7.Vertical polyacrylamide gel electrophoresis after silver staining,we can see control unsorted SUNE cells,CD133~+ cells and CD133~- cells on gel electrophoresis showed 6bp up the ladder,telomerase activity positive,and the ladder of CD133~- is shallower than control unsorted SUNE cells and CD133~+ cells,indicating that telomerase activity in the CD133~- is weakest,and ECV did not seen the 6bp ladder, telomerase activity negative.8.After transfecting hTERTp / TK / pGL,TK/pGL3 and CMV/TK/pGL3,we test telomerase activities again perform as TRAP-PCR silver staining of telomerase activity detection kit,the results are that telomerase activities of CD133~+ nasopharyngeal carcinoma stem cells in SUNE cell line after transfecting hTERTp / TK / pGL and CMV/TK/pGL3 decreased,the gray scale of their ladder strap on gel electrophoresis become shallower then before,but after transfecting TK/pGL3,there is no significant changes in telomerase activity.9.MTT resultsNasopharyngeal cancer stem cells:cell survival rate of hTERTp/TK/pGL3 group and CMV/TK/pGL3 group was significantly lower than control group,an average survival rate of cells in each group TK/pGL3 group,CMV/TK/pGL3 group,and hTERTp/TK/pGL3 group were 58.4±0.004%,20.5±0.004%,27.9±0.002%.namely, the effect to kill CD133~+ nasopharyngeal carcinoma stem cells in SUNE cell line in hTERTp/TK/pGL3 group and CMV/TK/pGL3 group was significantly higher than in TK / pGL3 control group,the effect of CMV/TK/pGL3 positive control group was significantly higher than hTERTp/TK/pGL3 group.Human umbilical vein endothelial cells ECV:cell survival rate of CMV/TK/ pGL3 group was significantly lower than hTERTp/TK/pGL3 group and TK/pGL3 group,CMV/TK/pGL3 group of cell survival rate was significantly lower than,an average survival rate of cells in each group TK/pGL3 group,CMV/TK/pGL3 group, and hTERTp/TK/pGL3 group were 57.7±0.001%,18.1±0.002%,58.2±0.001%. namely,the effect to kill human umbilical vein endothelial cells ECV in CMV/TK/pGL3 group was significantly higher than in hTERTp/TK/pGL3 group TK / pGL3 control group.Conclusion1.CD133 is one of surface markers for tumor stem cells from human nasopharyngeal carcinoma cell line SUNE. 2.Nasopharyngeal carcinoma stem cells do exist in nasopharyngeal carcinoma cell line SUNE.3.The human telomerase reverse transcriptase promoter/TK specific tumorspecific expressing system(hTERT/TK/pGL3) can specially kill CD133~+ nasopharyngeal carcinoma stem cells which is in nasopharyngeal carcinoma cell line SUNE,but do not kill the normal ECV cell.4.The human telomerase reverse transcriptase promoter/TK specific tumorspecific expressing system(hTERT/TK/pGL3) can reduce telomerase activity of CD133~+ nasopharyngeal carcinoma stem cells which is in nasopharyngeal carcinoma cell line SUNE.