Targeted Antitumor Effects Of Double Suicide Genes System Controlled By Specific Promoter In Multidrug-Resistant Glioma Cells

Objective:To establishment of a multidrug resistant C6/ADR cell line in vitro by stepwise selection in the presence of increasing concentration of Adriamycin and associated with high dose short explosure of ADM, and study on the morphological changes of C6 and C6/ADR cells and the detection of their characterization.Methods:(1) The C6 cells at logarithm growth stage were cultured in RPMI1640 medium with Adriamycin by successive exposure to increasing concentration of ADM (0.001, 0.01, 0.1, 0.25, 0.5μg/ml, each concentration for 4 weeks) in a fully humidified atmosphere containing 5% CO₂/95% air at 37℃. And then, after induced respectively with high dose short explosure of ADM (0.001, 0.01, 0.1, 1, 10, 100μg/ml) for 36h, MTT assay was used to detect the inhibition ratio of C6 cells with various concentration of ADM. The final concentration was 1μg/ml for the induction of C6/ADR cells next experiment.(2) The C6 cells at logarithm growth stage were plated in RPMI1640 medium added with 1μg/ml ADM by stepwise selection in the presence of increasing concentration of Adriamycin and associated with high dose short explosure of ADM. After 4 months, all of the cell lines were subcultured (one generation for 2 or 3 days) in RPMI1640 medium with 0.5μg/ml ADM. The C6/ADR were obtained from non-drug medium for 2 months and then used for detection by hematoxylin and eosin staining (HE staining) with light microscope and transmission electron microscope. (3) By using the MTT assay, the optical density (OD) was measured at 540 nm on an enzyme-linked immunosorbent assay reader with reference wavelength at 630 nm. And then, the growth curve was drawn with the OD as longitudinal coordinate and the growth time as abscissa to calculate the double time of C6 and C6/ADR respectively. The viability of cells was determined by comparing the number of viable cells. To analyze potential proliferation of single cell, clone formation assay was used to detect the reproductive activity of single cell. After performed with Giemsa staining (Sigma), cloning efficiency was determined by counting the number of colonies more than 50 cells as 1 positive colony in each well.(4) RT-PCR was performed to detect expression of MDR1 at the mRNA level in C6 or C6/ADR cells. Total RNA from C6 and C6/ADR cells was isolated using Trizol Reagent kit. One microgram of total RNA was then used in reverse transcription reactions with AMV-reverse transcriptase and Oligo dT18 non-specificity primer as described by the manufacturer. The resulting total cDNA was amplified by PCR. And then the amplified fragments were separated on 1% agarose gels for 30 min to observe on UV Transilluminator and be taken photo.(5) All the experimental data were gathered and put in order for statistical analysis as the statistically significant level of P-value less than 0.05.Results:(1) The abservation of light microscope demonstrated that the C6/ADR cells were the similar in morphology as C6 cells, including that the cell growth of both cells were more fast, and both appeared fibra and transparent without intracellular grains. The results of HE staining indicated that the characterization of nucleus was round, karyomegaly with apophysis, heterogeneity, and more were monocaryon, also appeared conjugate nuclei, besides there was mitotic figure with little endochylema. However, by using the transmission electron microscope, the observation result showed that there were lots of large and malformed nucleuses with major apophysis or microvillus, abundant endocytoplasmic reticulum and mitochondria in C6/ADR cells, compared with C6 cells.(2) By using MTT assay, the growth curve was drawn to display that the double time of C6 and C6/ADR was 59.52h and 64.58h respectively, which means that the reproductive activity of C6/ADR cells was not subjected to the multidrug resistance conspicuously. Moreover, the result of clone formation assay manifested that after induced with ADM, the influence on the reproductive activity of single cell of C6/ADR cells was not significant deviation comparing with C6 cells (P>0.05).(3) RT-PCR was performed to detect MDR1 mRNA levels to demonstrate that MDR1 was highly expressed in C6/ADR cells, while MDR1 mRNA levels were under detectable levels in C6 cells. Furthermore, the western blot was performed to confirm that the expression of MDR1 protein was only detected in C6/ADR cells, but not in C6 cells. In addition, flow cytometry was used to carry out quantitative analysis to show that the expression of P-gp was strongly positive in C6/ADR cells (42.51±7.82%), nevertheless, it was only 4.5% in C6 cells. It was proved that the multidrug resistance gene of multidrug-resistant cell was MDR1 gene, protein product of which was P-gp.Conclusion:(1) Between C6 and C6/ADR cells, there were no significant differences for observation in light microscope. However, through the transmission electron microscope, the ultrastructural organization was obviously different between them, especially abundant endocytoplasmic reticulum and mitochondria in C6/ADR cells, which demonstrated that following the increasing multidrug resistance, the synthesis of enzymes was enhanced endlessly, further indicating the complexity of multidrug resistance in C6/ADR cells.(2) After induced for MDR, there were no great differences in the cell doubing time and clone ability of single cell in C6/ADR cell compared with C6 cells, which illustrated that the reproductive activity and clone ability of single cell in C6/ADR cell were not subjected to the multidrug resistance conspicuously.(3) The multidrug resistant protein gene was strongly expressed in C6/ADR cells, with the product called P-glycoprotein (P-gp), whereas the expression of P-gp was not detected in C6 cells. Objective:To clone the multiple drug resistance protein (MDR1) promoter from C6/ADR, construct the double suicide genes expressive vector controlled by MDR1 promoter, and explore its targeted expression in C6/ADR cells.Methods:(1) Polymerase chain reaction (PCR) was used to amplify MDR1 promoter isolated from C6/ADR cells genomic DNA, which was linked with T vector. After cut by NdeI and HindIII, the A tailing was inserted into the 3'-end of MDR1 promoter fragments using TaKaRa DNA A-Tailing Kit. And then, this fragment was inserted into pMD 18Simple-T vector resulting in the pMD 18Simple-T-MDR1-Promoter vector construction. After the vector was transformed into JM109 competent bacterium, pMD 18Simple-T-MDR1P plasmid extracted from bacterium was amplified and confirmed through electrophoresis and sequencing.(2) The pcDNA3-TK (thymidine kinase) plasmid and MDR1P-T vector were digested with NdeI and HindIII enzymes, and MDR1 promoter was cloned into the upstream of pcDNA3-TK plasmid to exchange the CMV promote resulting in pcDNA3-MDR1P-TK vector construction, which was amplified by PCR and detected by direct sequencing. (3) Both pcDNA3-MDR1P-TK and pcDNA3-CD-TK were digested by BamH I, and then, the cytosine deaminase (CD) gene in pcDNA3-CD-TK plasmid was directly cloned into pcDNA3-MDR1P-TK plasmid to construct pcDNA3-MDR1P-CD-TK vector, which was confirmed through electrophoresis and sequencing.(4) This pcDNA3-MDR1P-CD-TK plasmid was transfected into C6 and C6/ADR cells respectively by liposome. After selection by 500μg/ml geneticin (G418), the transferred tumor cell lines were stably established. Then these cell lines were examined through PCR, and RT-PCR to respectively detect the integration and expression of TK and CD genes in C6 and C6/ADR cells.Results:(1) The 260bp MDR1 promoter fragment isolated by PCR from the C6/ADR cells was identified as the expected fragment from the C6/ADR cells. After cloned into T vector, the MDR1 promoter fragment amplified was confirmed by direct sequencing reactions. The result showed that the MDR1 promoter, of which the sequencing was the same as the gene bank, had been cloned successfully into pMD 18Simple-T vector.(2) The pcDNA3-MDR1P-TK was constructed successfully by using molecular cloning technique after MDR1P was inserted into pcDNA3-TK. The result of PCR demonstrated that the 1600bp PCR product of pcDNA3-MDR1P-TK recombinant plasmid was detected by using electrophoresis. Furthermore, CMV promoter replaced with MDR1 promoter (remaining enhancer sequence partly) was confirmed by direct sequencing, meanwhile the sequence of plasmid has been proved to be correct.(3) After enzyme digestion with BamH I and purification, CD fragment was cloned successfully into the upstream of TK gene in pcDNA3-MDR1P-TK plasmid to construct pcDNA3-MDR1P-CD-TK. The PCR indicated that the lane 1-4 was positive clone, which meaned that the recombination was proved to be correct initially. At the same time, the result of sequencing confirmed that the sequence of this fragment from this plasmid was the same as the gene bank and the recombination was right.(4) After stably transferring double suicide genes controlled by MDR1 promoter into C6 or C6/ADR cells, two transfectants were called differently for C6/CDTK and C6/ADR/CDTK, which were similar to the previous C6 and C6/ADR cells. PCR indicated the double suicide genes were integrated into C6 and C6/ADR cells. By using RT-PCR, the result revealed that CD and TK genes expressed in C6/ADR/CDTK cells, whereas not in C6/CDTK cells.Conclusion:(1) The CD-TK fusion sucide genes expressive vector controled by MDR1 promoter has been constructed successfully.(2) The pcDNA3-MDR1P-CD-TK has been transferred successfully into C6 and C6/ADR cells respectively to obtain stable C6/CDTK and C6/ADR/CDTK cells.(3) It has been proved that the CD-TK fusion sucide genes expressive vector controled by MDR1 promoter was targeted expression in MDR1 positive cells (C6/ADR/CDTK cells), whereas not in C6/CDTK cells. Objective:To investigate the targeted killing effects of cytosine deaminase (CD) -thymidine kinase (TK) fusion suicide gene/prodrug therapy (GCV+5-FC) controlled by multidrug resistance gene promoter on MDR glioma cells.Methods:(1) Transfected C6 and C6/ADR cells were divided respectively into two groups: one group treated with prodrug and another group without prodrug. They were plated respectively in a 6-well plate and maintained in RPMI1640 medium with 60μg/ml ganciclovir (GCV) and 600μg/ml 5-fluorocytosine (5-FC). After cultured for 24h, these cells were observed by using inverted microscope.(2) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at logarithm growth stage were maintained respectively in RPMI1640 medium with various concentrations of GCV and/or 5-FC compared with control groups without prodrugs. By using the MTT assay, the OD value was measured at 540 nm on an enzyme-linked immunosorbent assay reader with reference wavelength at 630 nm to calculate the cell survival rate. And then, the cell survival curve was drawn with the cell survival rate as longitudinal coordinate and the prodrug level as abscissa to observe the cell survival state. (3) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at logarithm growth stage were maintained respectively in RPMI1640 medium with or without 60μg/ml GCV and 600μg/ml 5-FC. The cell apoptosis in different phase of the cell cycle were measured by flow cytometry in these cells and TUNNEL assay was used to detect the cell apoptosis.(4) The C6, C6/ADR, C6/CDTK and C6/ADR/CDTK cells at 3×10⁴/ml were maintained respectively in RPMI1640 medium with or without 60μg/ml GCV and 600μg/ml 5-FC. The clone formation assay was used to detect the reproductive activity of single cell. After performed with Giemsa staining (Sigma), cloning efficiency was determined by counting the number of colonies more than 50 cells as 1 positive colony in each well.(5) All the experimental data were gathered and put in order for statistical analysis as the statistically significant level of P-value less than 0.05.Results:(1) After the addition of prodrugs, the light microscope demonstrated that the growth of untransfected C6 and C6/ADR cells was in good condition, and there were no obvious changes of cell population and cell proliferation, furthermore, the change with the combination of GCV and 5-FC was no greater than with single prodrugs; however, the cell endoparticle of C6/ADR/CDTK increased gradually, cell morphous were shrinking, the ability of adherence became weak, the growth condition went bad and the cell number decreased, especially treated with both GCV and 5-FC.(2) The MTT assay demonstrated that the cell survival rate in C6/CDTK was slightly decreased compared with C6 cell following increasing drug concentration; the cell survival rate in C6 and C6/ADR cells was similar with no marked change; nevertheless, compared with others, the inhibition of cell proliferation in C6/ADR/CDTK was more predominance and the cell survival rate was reduced significantly (P<0.05), which meaned that both GCV and 5-FC had an obvious killing effect on C6/ADR/CDTK cells in a concentration-dependent manner.(3) The flow cytometry was used to manifest that compared with C6/CDTK cells, the rate of cell apoptosis in C6/ADR/CDTK cells was increased conspicuously after the addition of prodrugs; moreover, the effect of the combination of GCV and 5-FC was more significant than single prodrug, especially the rate of cell apoptosis reaching up to 22.64% (P<0.05); in addition, the cell cycle of C6/ADR/CDTK stayed in G1-stage without DNA synthesis. At the same time, the TUNNEL assays replayed that the result of C6/ADR/CDTK was positive and others were negative.(4) The clone formation assay expressed that cloning efficiency of C6, C6/ADR and C6/CDTK cells went up to 90% above, but the cloning efficiency degraded significantly in C6/ADR/CDTK cell with increasing prodrug concentration. And also, the cloning efficiency was more conspicuously decreased with combination of both prodrugs than single prodrug, only was 16.71±0.31%.Conclusion:The double suicide genes system controlled by the MDR1 promoter was transfected successfully into multidrug-resistance (MDR) and non-multidrug-resistance glioma cells which were treated with GCV and/or 5-FC prodrug. The results showed that the MDR1 promoter driven CD-TK fusion double suicide gene/prodrug system had a significant antitumor effect in the multidrug-resistance cells (C6/ADR cells) compared with non-multidrug-resistance glioma cells (C6 cells), especially that prodrugs in combination could have a more powerful killing effect than single prodrug. In conclusion, this study indicated that the suicide gene therapy controlled by the specific promoter had a selective and specific killing effect on MDR glioma cells, which was proved to be targeted effectiveness of this method. We can believe that this study will provide a sound basis for targeted gene therapy for multidrug resistance glioma.