In Vitro Study Of Lethal Effect Of TK/CD Fusion Suicide Gene Under The Control Of PCEA-CMVE Chimeric Promoter On Different CEA Expression Level Lung Cancer Cell Lines

BACKGROUND: Lung cancer is the most rapidly developed cancer in terms of morbidity and fatality rate. Every year there are more than one million people die from lung cancer. Conventional therapies include surgery, chemotherapy, radiotherapy etc, have no progress on the five-year survival rate. We need a more direct, safe and effective way to treat the disease. Gene therapy offers a new therapeutic mode to treat the lung cancer. A series of researches have shown that the combination of gene therapy and conventional therapies can improve the therapeutic effect. Gene transfer therapy introduces ectogenic genes into the cancerous cells or the surrounding tissue to inhibit the cell proliferation and induce the cell death. Because of the high effective and low toxic, suicide gene therapy has become an important part and a hot topic of gene transfer research. At present, the most mature research of suicide gene systems are HSV-TK/GCV and CD/5-FC. Because of the medicine resistance with one suicide gene and the different medicine sensitivity of different tumor cells, drug combination can decrease the resistance and increase the curative effect. To target the tumor cells, we can use the tumor specific regulatory element to control the suicide gene express only in the tumor tissue, for the specific lethal effect. We have successfully built the recombinant vector of CMVE-pCEA-TK-IRES-CD, including the CEA promoter, and target the CEA positive lung cancer cells through driving the downstream gene HSV-TK/CD to educe the lethal effect. We choose the lung cancer cells with different expression level of CEA, to detect the targeted effect of CMVE-pCEA-TK-IRES-CD. At the same time, detect the cytoxicity and apoptosis through the interference of prodrug GCV and 5-FC, compare the variability of lethal effect in lung cancer cells wiith different CEA expression level and the synergetic effect of double suicide gene.Note: the study is sponsored by the nation's natural science foundation. (No.30471997)OBJECTIVE: Through the in vitro study of lethal effect of TK/CD fusion suicide gene on different CEA expression level lung cancer cell lines, confirm the target characteristic of CMVE-pCEA-TK-IRES-CD carrier system and detect the sensitivity of prodrug GCV and 5-FC, offer experimental data for the clinical trial.METHODS: Use the method of immunohistochemisty, reverse transcription PCR and real time PCR to confirm the expression level of CEA in A549, SPC-A-1, NCI-H520, 16HBE cells. Use the EGFP, lipofectamine 2000 to obtain the best condition of transfection effective, then use the best condition to transfect the lung cancer cells. To transfect the recombinant vector CMVE-pCEA-IRES-EGFP to the different CEA expression level cell lines for detecting the targeted characteristic through the EGFP. After that, transfect the CMVE-pCEA-TK-IRES-CD to the A549 and SPC-A-1, use the G418 to screen the stable resistant clone. Through the real-time PCR to determine the relatively quantitate of TK and CD gene. Use the WST-1 to detect the lethal effect of A549/TK+CD and SPC-A-1/TK+CD with single or combine medication, compare with the A549, SPC-A-1, NCI-H520, 16HBE cells. Mix the A549 and A549/TK+CD cells with Different percentage to assay the bystander effect(BSE), so do the SPC-A-1 and SPC-A-1/TK+CD cells. Finally, observe the apoptosis and the cell cycle change by flow cytometer and ultra-structure by electron microscope.RESULTS: Immunohistochemisty, reverse transcription PCR and real time PCR confirm that A549, SPC-A-1 are CEA positive cells and NCI-H520, 16HBE are CEA negative cells. Otherwise, the CEA level of A549 is higher than the SPC-A-1. At the meanwhile, when we transfect the CMVE-pCEA-IRES-EGFP to these two cell lines, the expression of EGFP is related to the expression level of CEA. All the lung cancer cells can reach the transfection effect of 70% and the 16HBE cell is about 54%. CMVE-pCEA chimeric promoter can only promote the downstream gene TK/CD express in the CEA positive cells(A549 and SPC-A-1). Through the screen of G418, reverse transcription PCR certifies the TK/CD gene has been transfected into the targeted cells. To the A549/TK+CD and SPC-A-1/TK+CD cells ,the 5-FC and GCV have the significant cytoxicity, with the inhibitory concentration 50(IC50) of 624.7996μg/ml , 1713.6470μg/ml和169.8896μg/ml,638.7062μg/ml.There are about 1.56,1.59 and 4.23,2.46 times compare with the non-transfect cell A549 and SPC-A-1. Combine the 5-FC and GCV has the significant synergetic effect. The coeffeicient of drug interaction (CDI)was lower than 1. The apoptosis and cell cycle observed by flow cytometre are related with the density of prodrug 5-FC and GCV and the modality of single or combine use. Cells present the apoptosis change in ultra-structure by electron microscope.CONCLUSION: Comfirmed the expression level of CEA in 4 kinds of lung cancer cells. In A549 and SPC-A-1, the level of EGFP is related to the level of CEA. CMVE-pCEA chimeric promoter can only promote the downstream gene TK/CD express in the CEA positive cells(A549 and SPC-A-1). The prodrug 5-FC and GCV have the more significant cytoxicity in A549/TK+CD and SPC-A-1/TK+CD cells, they can induce the obviously apoptosis and inhibit the cell proliferation, combined use has the synergetic effect. This experiment offers the useful data to study the therapeutic effect of fusion gene TK/CD in the lung cancer patients of CEA positive.