The Changes of Cell Cycle Regulatory Proteins Expression by AdCMVp53 in Cervical Cancer Cell Lines / 대한산부인과학회잡지

OBJECTIVE: In this study, we investigated the cell growth inhibition, regulation of cell cycle and induction of apoptosis through recombinant p53 adenoviral vector delivery into cervical cancer cell line SiHa, to explore the possibility of p53 gene therapy. METHODS: We infected SiHa with AdCMVp53 at 50 MOI. After 48 hours, the regulation of cell cycle and apoptosis were investigated with FACS. The gene expression profiling associated with cell cycle was also investigated with cell cycle DNA membrane chip. RESULTS: SiHa cells were arrested in the G1 phase by AdCMVp53 and showed cell growth inhibition via apoptosis. The gene expression profiles involved in cell cycle including cyclin-dependent kinase inhibitor 1C (p57, Kip2), RAD9 (S.pombe) homolog, and MAD2 (mitoticarrest deficient, yeast, homolog)-like 2 were up-regulated by more than three-fold, as compared to control group. In contrast, 6 genes such as retinoblastoma-like 2 (p130), and cyclin H were down-regulated by more than three-fold. Several genes known as being differentially up- or down-regulated compared to control were confirmed by RT-PCR and Western blotting assays. CONCLUSION: The adenoviral p53 gene delivery into cervical cancer cell line, suggesting the possibility of p53 gene therapy in cervical neoplasia make the cell growth inhibition and changes of cell cycle-associated gene expression.

Adenovirus and Lipofectin Mediated p53 Gene Transfection Efficiency in Human Cervical Cancer Cell Lines and Xenografted Nude Mouse / 대한산부인과학회잡지

BACKGROUND: The classical treatment of the cervical cancer is surgery, radiotherapy, chemotherapy. Even though the improvement of treatment successful rate, conventional therapy has some limitations. Recent cutting edge of cancer therapy has been developed in gene level including understand the biological characteristics of the cancer cells, enhance the human immune response, suppress the cancer cell proliferation. Therefore, the gene therapy is proposed to new treatment strategy. PURPOSE: The transfection efficiency of cervical cancer cell lines and cervical cancer cell line xerografted nude mouse was investigated by transfection of liposome and infection of adenovirus mediated suppressor(p53) and reportor(LacZ) gene. METHOD: The cervical cancer cell lines was used in this study were CaSki, SiHa (HPV16 positive, wild type p53 gene), HeLa, HelaS3(HPV18 positive, wild type p53 gene) and C33A, HT3(HPV negative, mutant p53). Direct plasmide and AdCMVp53 gene transfection was performed by using liposome system (pRcCMVLacZ / lipofectin, FuGene 6, Ca-phosphate). LacZ gene was used as the reportor gene for the transfection efficiency evaluation. Expression of p53 in cell lines and tumor tissue was confirmed by western blot and immunohistochemical staining. Xenografted nude mouse of SiHa cell line was infected by AdCMVp53 and AdCMVLacZ. Transfection efficiency was observed by same as above. RESULTS: In cervical cancer cell lines, gene transfection using liposome system(pRcCMVLacZ/lipofectin, FuGene 6, Ca-phosphate)revealed different transfection efficiency, especially pRcCMVLacZ in Fugene 6 showed 18-40% of high transfection efficiency in 6 cervical cancer cell lines by X-gal staining and AdCMVp53 showed 95-98% of the high transfection efficiency in HeLa, C33A. AdCMVp53 was significantly expressed at 2-5days after injection xenografted nude mouse on the western blot and transfection efficiency was 19.79+/-5.36, 26.26+/-11.69, 14.77+/-3.98,15.99+/-6.43%(day1-5). AdCMVLacZ were found to immuno- histochemistry analysis, in vivo transfection efficiency was 61.26+/-4.66,59.63+/-9.12, 29.46+/-14.33, 31.73+/- 22.64%(day 1-5) atx200 and 88.65+/-8.65, 70.85+/-20.94, 40.75+/-25.44, 48.21+/-10.97% (day 1-5) atx400. CONCLUSION: As a results, adenovirus-mediated transfection efficiency was higher in vivo experiment compared to cell lines. These high efficiency of adenovirus-mediated suppressor gene(p53) could become a significant meaningful data gene therapy strategy both transgenic mice and cervical cancer cell lines.

Development of Gene Therapy Strategy Using Plasmid and Adenovirus in Cervical Cancer Treatment / 대한산부인과학회잡지

BACKGROUND: The basic treatment of malignant tumors is surgery, radiotherapy, chemotherapy. Even though, the object of these treatments is to kill cancer cells, they have limitations. So, in future studies of treatment of cancer, we should look into increasing human immune response using gene therapy in order to induce damage to tumor cells. OBJECTIVE: The cell growth inhibitory effect of cervical cancer cells was investigated by direct transfection using liposome(pRcCMVp53/lipofectin). and by indirect transfection using Adenovirus(AdCMVp53). METHODS: The cervical cancer cell lines we used in this study were HPV16 positive, having inhibitory gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3, LacZ gene was used as the marker gene for the transfection efficacy. Direct transfection was done by using lipofectin (pRcCMVp53/lipofectin) and indirect transfection was done by using virus, AdCMVp53. The effect of tumor cell growth inhibition was measured by cell counting assay. RESULT: Inhibition of growth of cervical cancer cells in cell counts of direct transfection was CaSki(88.5%), SiHa(59.1%), HeLa(86.0%), HeLaS3(78.0%), C33A(91.3%) and HT3(74.0%). Inhibition of growth of cervical cancer cells in cell counts of indirect transfection was CaSki(97.4%), SiHa(91.6%), HeLa(95.8%), HeLaS3(99.7%), C33A(97.3%) and HT3(87.4%). CONCLUSION: The inhibition of cell growth of cervical cancer cells by direct and indirect transfection was significantly reduced, and showed little differences depending on the type of cells. These results will have a great meaning in treating cervical cancer patients using gene therapy by direct or indirect transfection