| CDK4 is a member of cyclin-dependent kinases (CDKs) family. It plays an important role in controlling cell cycle. The CDK4 gene has been confirmed as an oncogene. Amplification and over-expression of CDK4 has been found in tumorigenesis of several types of human cancer. It becomes a potential target for the treatment of human diseases in which the control of cell proliferation is deregulated.At present, there are several other methods to inhibit biological activity of intracellular molecules. RNA based therapies, such as ribozymes, antisense RNA or RNAi are less stable and can only be used against RNA. Other protein-based therapies, such as dominant negative inhibitors, rely on competition with a native binding species and therefore require excessive concentration. In addition, these targets generally are proteins. Whereas intrabodies, by linking appropriate intracellular trafficking signal sequences to their coding genes, can be directed to all intracellular compartments including cell nucleus, cytoplasm or other cell organs. Recently intrabodies have been studied in terms of treatments for tumor growth and even clinical trials for cancer. As potential direct therapeutics, intrabodies are distinguished from small-molecule and peptide drugs in their enhanced target binding specificity and stability. Thus intrabody technique may become a pioneer of a new approach for cancer therapy.In present study, an expression plasmid pNLS-AK including a nuclear localization signal (NLS) and scFv gene against human CDK4 was constructed. In order to test the expression and activity of pNLS-AK, MCF-7 cells and HeLa cells were transfected with pNLS-AK and pcDNA3.1, respectively. Then expression and subcellular distribution of NLS-AK was detected by RT-PCR, Western-blot, co-immunoprecipitation and fluorescence staining analysis. At the same time, MTT analysis was used for cell proliferation analysis. The cell cycle and apoptosis changes of the stably transfected cells were detected by Flow Cytometry.Fluorescence microscope analysis demonstrated that both MCF-7/pNLS-AK and HeLa/pNLS-AK cells showed positive staining in cell nucleus, but the MCF-7, HeLa, MCF-7/pcDNA3.1 and HeLa/pcDNA3.1 cells did not show any fluorescence staining. RT-PCR analysis demonstrated that the NLS-AK mRNA was expressed only in MCF-7/pNLS-AK and HeLa/pNLS-AK cells, not in MCF-7, HeLa, MCF-7/pcDNA3.1 and HeLa/pcDNA3.1 cells. Western-blot analysis showed that NLS-AK was detected only in MCF-7/pNLS-AK and HeLa/pNLS-AK cells, but was not detected in MCF-7, MCF-7/pcDNA3.1, HeLa, and HeLa/pcDNA3.1 cells. Co-immunoprecipitation analysis indicated that only MCF-7/pNLS-AK and HeLa/pNLS-AK cells can develop a specific band with molecular weight of 34 kD. It implied that the NLS-AK protein can expression and integrate with CDK4 inside the cells. All these results suggested that NLS-AK gene was already transferred into MCF-7 and HeLa cells and was successfully expressed.The growth curve showed that the expression of NLS-AK inhibited the growth of MCF-7 cells and HeLa cells significantly(p<0.01). Cell cycle analysis by FCM showed that the expression of NLS-AK arrested the cell cycle of MCF-7/pNLS-AK and HeLa/pNLS-AK cells at the G1 phase, inhibiting the cells to enter the S phase of MCF-7/pNLS-AK and HeLa/pNLS-AK cells. Cell apoptosis analysis indicated that expression of NLS-AK induced distinctly MCF-7/pNLS-AK and HeLa/pNLS-AK cells apoptosis.In conclusion, stable expression of NLS-AK regulated cell cycle and cell proliferation of MCF-7/pNLS-AK and HeLa/pNLS-AK cells. These data strongly suggested that intracellular scFv may become an effective approach for tumor gene therapy.
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