| The development of tumor often correlates with the chaos of cell cycle. Some key regulators of cell cycle can be the potential target of tumor gene therapy. The regulation of G1/S transition in cell cycle is especially important. Because in this point, the cell will be determined to proliferate continuously or enter the G0 phase, or lead to the terminal differentiation or to death. CyclinD1 and p16, as the positive and negative regulators, play an important role in this progression. CyclinD1 can activate CDK4/6 by integrating with it, and promote the transition of G1/S.Whereas p16 inhibits the activity of CDK4/6 by integrating with, competitively with Cyclin D1. Therefore CyclinD1 and p16 always keep balance with each other in common. While in tumor cells, there are always the over-expression of Cyclin D1 and the extensive inactivity of p16, and in most cases, this two abnormalities will exist simultaneous. So they can be the targets of gene therapy. Investigators injected the anti- Cyclin D1 antibody, or the antisense oligonucleotide, or some plasmids with the aitisense Cyclin D1 into tumor cells , and found that these could improve the phenotype of transformed cells, and inhibit the proliferation of tumor cells. Some researchers have demonstrated that the transfection of adenovirus-mediated p16 gene could achieve good antitumor effect in breast cancer, ovarian cancer, lung cancer, esophageal cancer, melanocytoma, pancreatic carcinoma, and so on.For the tumorigenesis is a complex progression with many genes participating, it's important and necessary to develop multi-gene combination therapy. As mentioned above, the over-expression of Cyclin D1 and the inactivity of p16 often exist simultaneous, and their downstream responders are CDK4/6. So the inhibition of CyclinD1 in combination with the restoration of p16 may strengthen the inhibition to CDK4/6. Their combination can be an available gene therapy strategy.Intrabody is a new phenotype knock-out technology based on antibody engineering and cellular signal transduction. In our previous work, we have constructed an eukaryotic expression vector with a 9E gene, coding an endoplasmic reticulum (ER)-retained intrabody against CyclinD1. In our study, we transfected the 9E gene and the wide-type p16 gene into the MCF-7 cells, in whichCyclinD1 is over-expressing and p16 is homozygosis deletion, respectively or in combination. We use RT-PCR, Western-blot, co-immunoprecipitation and indirect immunofluorescene to detect their expression in cells. At the same time, we analysed their single and combined antitumor effect by FACS, MTT-cell proliferation analysis and DNA ladder.The follows are results:1. The expression of p16 and 9E in MCF-7 cells transfected with p16 and 9E, respectively or in combination.In order to detect the expression of p16 and 9E in tumor cells, we first analysed the MCF-7 cells transfected transiently with p16 and 9E, alone or in combination. Their expression in the level of transcription was detected by RT-PCR with their specific primer. Western-blot results showed that, the specific bands with the molecular weight of 16 kD and 31 kD when using specific anti-p16 and anti-E-Tag monoclonal antibody as primary antibody. The indirect immunofluorescene also showed that the p16 was expressed in the whole cell, while 9E was expressed primarily in the cytoplasm.In the latter, we carried out the stable transfection of p16 and 9E into MCF-7 cells alone or in combination to analyse their antitumor effects. Western-blot and indirect immunofluorescene suggested that the strains transfected stable screened were positive clones, in which there were the expression of p16 and 9E except the cells with the transfection of the control vector. They were named MCF-7/plpBg, MCF-7/plpBg-p16, MCF-7/plpBg-9E, MCF-7/plpBg-p16+9E, respectively.2. The integration of intrabody 9E with CyclinD1 in MCF-7 cellsIn order to detect whether 9E could integrate with CyclinD1 inside the cell, we did the co-immunoprecipitation of 9E using anti-E-Tag antibody was conducted. After the cells lysis was extracted, anti-E-Tag monoclonal antibody was added into the lysate, incubated for 1-2h, and then adds Protein G-Sepharose into the supernatant, mixed, and then was detected by Western-blot with anti-CyclinD1 polyclonal antibody as primary antibody. The result showed that there was a specific band with molecular weight of 34 kD, that's CyclinD1 protein. These suggested that 9E still held the ability of integrating with CyclinD1 inside the cell.3. The strengthened antitumor effects by the transfection of p16 and 9E in combination. We analysed the cell cycle, apoptosis, and the cell proliferation of MCF-7 cells transfected with p16 and 9E, alone or in combination, by FACS, MTT-cell proliferation analysis and DNA ladder. We found that after the transfection of p16 and 9E, the percentage of G1 phase increased from 41.14% (Mock) to 59.52%(MCF-7/plpBg-p16),59.76%(MCF-7/plpBg-9E)and 79.18%(MCF-7/plpBg-p16+9E), respectively. The percentage of apoptosis also increased from 2.47% (Mock) to 13.15% ( MCF-7/plpBg-p16 ), 12.31%(MCF-7/plpBg-9E)and 17.13%(MCF-7/plpBg-p16+9E), respectively. These results showed that the transfection of p16 and 9E alone or in combination resulted in G1 arrest and induction of apoptosis, and the effects of co-transfection of p16 and 9E were more significant. MTT-cell proliferation analysis also demonstrated the transfection of p16 and 9E alone or in combination could inhibit cell proliferation, and the effects of co-transfection of p16 and 9E were more significant.In conclusion,p16 and 9E were expressed in MCF-7 cells transfected with p16 and 9E, respectively or in combination. Their expression also induced obvious antitumor effects, and the combination transfection of them was more significant than single transfection. These results provided new theoretical and experimental evidence for gene therapy, especially for combined gene therapy.
|
PDF Full Text Request