Screening Cancer Stem Cells Based On The CD133/Prominin1Promoter Activity

Based on the differences of CD133/Prominin1gene promoter activity in tumor cells,we have screened pCD133(+) U251cells (with CD133gene promoter activity) and pCD133(-) U251cells (without CD133gene promoter activity) through the regulation of neomycinresistance gene and herpes simplex virus-thymidine kinase gene specific expression byCD133gene promoter. By comparing the differences of CD133expression and biologicalfunction on surface of two sorts of cell population, we created a new cancer stem cell sortingmethod according to differences of CD133gene promoter activity in tumor cells. The resultsin this study were as follows,1. We described the differences of CD133gene promoter activity in glioma cell lineU251.When U251cells were infected by the lentiviral vector packaging particles of CD133promoter regulating green fluorescent protein gene expression, green fluorescent proteinexpression was observed in a small part of U251cells, indicating that the CD133promoteractivated only in a small part of the U251cells. This provided theoretical basis for the tumorstem cell screening through promoter activity differences.2. pCD133(+) of U251cells were successfully screened from the U251cells.U251cells were infected by the lentiviral vector packaging particles of CD133promoter regulating neomycin resistance gene expression, after G418selection, the cellswith CD133promoter activity survived, while the cells without CD133promoter activitywere killed. pCD133(+) U251cells could be screened from the tumor cell population, ableto continue growing when G418given.3. pCD133(-) of U251cells were successfully screened from the U251cells.U251cells were infected by the lentiviral vector packaging particles of CD133promoter regulating herpes simplex virus-thymidine kinase gene expression,after hygromycin selection and ganciclovir given, the cells with CD133promoter activitywere killed, while the cells without CD133promoter activity survived. pCD133(-) U251cells could be screened from the tumor cell population.4. We illustrated the cells number with surface marker CD133in the two cells.By RT-PCR and immunocytochemistry to detect CD133protein expression of two cells,it showed that there were80%CD133(+)and20%CD133(-) cells in pCD133(+)U251cells.There is no CD133positive expression in pCD133(-)U251cells. This showed that by this method CD133(+) cells couldn't be fully isolated but CD133(-) cells could be effectivelyisolated from tumor cells.5. We illustrated cell proliferation differences of two cell types.By cell growth curves and cell cycle, they showed that pCD133(+) cells had strongerproliferation than pCD133(-) cells in serum-containing culture conditions. Results of clonegeneration capacity experiment showed that pCD133(+) cells colony formation (average65clones/100cells) was significantly higher than pCD133(-) cells(without clone generationcapacity).6. pCD133(+) cells could form pCD133(-) cells, while pCD133(-) cells could notform pCD133(+) cells.By flow cytometry, changes of CD133protein expression on cell surface were detectedduring the course of pCD133(+) cells without G418given and pCD133(-) cells withoutGCV given for1day,6days,12days. The results showed that CD133protein expression ofpCD133(+) cells gradually decreased, while that of pCD133(-) cells did not change. Itindicated that pCD133(+) of U251cells could gradually differentiate into CD133(-) cellsunder the conditions without drug pressure and in serum-containing culture conditions.After pCD133(+) cells and CD133(-) cells were infected by lentiviral vectorpackaging particles of CD133promoter regulating green fluorescent protein gene expression1day,6days,12days respectively, green fluorescent protein expression was detected by flowcytometry. The results showed that green fluorescent protein expression in pCD133(+) cellsgradually reduced, while there was no change in pCD133(-) cells. It indicated that CD133promoter activity gradually lost when pCD133(+) cells were cultured without drug pressureand in serum-containing culture conditions.From above mentioned research results, we illustrated the application of CD133promoter activity differences in cell population in order to screen tumor stem cellseffectively for the first time. Scientific significances are:1. Through this method, the cells with cell surface markers CD133(-) can bescreened successfully.The cells without CD133promoter activity couldn't carry out CD133gene transcriptionand translate into CD133protein, therefore the screening CD133(-) cells were sure to beCD133(-) cells. It could avoid the false-negative (there are CD133(+) cells in CD133(-) cells)for antigenic epitope differences by using CD133antibody. The results of study on CD133(-)biological activity also confirmed that only the CD133(+) cells had cancer stem cell properties.2. Further confirmed the inconsistency of the transcription activity and CD133surface marker of CD133gene.Majority studies have shown that CD133expression of the U251glioma cell surface incell groups usually is2%-5%, and the result of our study by flow cytometry was about3%.The CD133promoter activity in cells accounted for about15%, significantly higher thanCD133expression on cell surface. The results of flow cytometry showed that81%(not allcells) of the cells expressed CD133positive. This indicated the inconsistency of thetranscription activity and CD133surface marker of CD133gene.3. In this study, the screened cancer stem cells could be amplified in adherentculture conditions.In most reported, the glioma stem cells, including tumor cells, were often suspendedamplified in serum-free culture. In this study, CD133(+) cells could be effectively amplifiedin serum-containing culture conditions when resistance drugs were given.4. In this study, the research results suggested that target killing tumor stem cellswith the CD133promoter activity was superior to target cell surface CD133protein asa target for treatment.The tumor cells with CD133promoter activity include CD133(+) and CD133(-) cells,so targeted cancer therapy can be completely targeted CD133(+) cells. However, due to thevariation of the protein, some target therapy is difficult to get rid of the cells of CD133expression using CD133protein on cell surface as a target, thus affecting the effects oftreatment.5. Our research techniques and theory can be extended to screen other cancer stemcells with CD133sorting signs. For the reason of the specificity of CD133promoter geneexpression in different tissues, isolation of cancer stem cell should choose a differentpromoter depending on the type of tumor cells.Although we have effectively screened tumor stem cell through the differences ofCD133promoter activity in different cell groups in vitro, tumorigenicity capacity should befurther verified in animals. We should further study in vivo whether targeting clearanceCD133(+) cells could effectively inhibit tumor growth and metastasis after cells infectedwith the suicide gene and drug given, in order to further improve basis for tumor stem celltheory.