Study On The Activity Of Carcinoembryonic Antigen Promoter

Cancer is a kind of malignant disease which threatens for the people's health. The study of the form, prevention and therapy of cancer were, is and will be the hot point of the study in life sciences for a long time. The most common therapy method of cancer now is resection, radiotherapy and chemotherapy are also used as assistant, while they have serious side effect. Therefore, it is important and necessary to study on the progression of cancer, and new methods and technologies in the cancer gene therapy.Selectively transfer system plays an important role of cancer gene therapy. The main difficulty of cancer gene therapy is how to kill the tumor cells selectively. It is necessary and urgent to find effective selectively transfer systems.Carcinoembryonic antigen (CEA) is the first tumor specific antigen to be isolated and identified, and it is associated with many kinds of tumors. Therefore, CEA is an ideal material to be used for the selectively transfer system. We have study on the possibility of using the CEA promoter in specific cancer gene therapy. We constructed a eukaryotic probe vector pEGFP-Nd and a recombinant expression plasmid pCEA-EGFP used enhanced green fluorescent protein (EGFP) as reporter gene, which was driven by the CEA promoter.After transfection with the plasmid pCEA-EGFP into different tumor cells, we found that the CEA promoter can drive the report gene EGFP expressing in the CEA positive colorectal adenocarcinoma cells LS-174T, SW480 and lung cancer cells A549, while no expressing in the CEA negative cervix cancer cells HeLa and larynx cancer cells HEp-2. As contrast, the control plasmid pEGFP-N1, whose reporter gene EGFP is driven by the CMV promoter, has expressed in all the cells above, show no selective activity at all. The result showed that the CEA promoter could be used in the selective gene therapy for CEA positive cancer cells. The results of flow cytometer are consistent with the results of fluorescent reversemicroscope. However, we have made a comparison of the statistical data, which showed that the selective activity of the CEA promoter in the three CEA positive cells became weaker from SW480. LS-174T to A549.We constructed a recombinant plasmid pCEA-bak using bak as effecter gene, which was driven by the CEA promoter. After transfection with the plasmid pCEA-bak into different tumor cells, we found that it can induce apoptosis in the CEA positive cells SW480, while no apoptosis in the CEA negative human cells HeLa. As contrast, the control plasmid pcDNA3-bak, whose effecter gene bak is driven by the CMV promoter, has induced apoptosis in both cells, show no selectivity at all. The result showed that the CEA promoter could drive bak gene to induce apoptosis in CEA positive cells selectively. It is promised to be used in the selective gene therapy for CEA positive cancers after improved.Bel 10 is a newly discovered cytokine, and it plays an important role in both apoptosis and anti-apoptosis. These special characters show that it must play a very important role in the cell physiology. It may be a key molecule in the cellular signal pathway. CEA is the most common tumor antigen, its tumor specificity maybe have some relation with certain important trans-acting regulate elements.Miss Cheng Ming, one Master student in our lab, had proved that Bel 10 could active the transcription of leu and lacZ in yeast two hybrid. In order to study its function and prove the interaction of Bel 10 and CEA promoter, we constructed a plasmid pCMV-EGFP and co-transfected it with the plasmid pCEA-EGFP, which contains the reporter gene EGFP driven by the CEA promoter, into the CEA negative HeLa cell. We found that Bel 10 can activate the CEA promoter and so caused the expression of EGFP. While the HeLa cells transfected with pCMV-EGFP or pCEA-EGFP alone had no expression of EGFP. We expressed and purified the Bel 10 protein and transferred to nitrocellulose after electrophoresis, it showed negative result in Northern Blot with DIG labeled CEA promoter. While after incubated Bcl10 with the e...