Effects Of Adenovirus Type 5 E1A Gene On Proliferation And Metastasis Of Human Lung Adenocarcinoma Cells

Lung carcinoma is a common malignant tumor in our country whose morbidity is increasing rapidly in recent years. The initiation and progression of lung cancer are involved in many oncogenes and tumor suppressor genes, such as the deletions of genetic material of 2q and 3q, the over expressions of HER-2/neu gene, the amplification of c-myc gene, the inactivation of function of p53 gene etc, which makes gene prevention and gene therapy possible . The Ad5E1A gene transcribes two mRNAs of 12S and 13S, and codes for two major related proteins of 243 and 289 amino acids by differently splicing. The E1A proteins are nuclear phosphoproteins that are multifunctional transcription factors. The E1A products are positive and negative regulators for the transcription of many cellular genes. At present, non-small cell lung cancer is less as the subject to the study of E1A gene than other. The aim of study is to research the effects of Ad5E1A gene on proliferation and metastasis of the human lung adenocarcinoma (Anip973). The results as follows:1. Construction of the mammalian expression recombinant pcDNA3-E1A.A E1A gene recombinant expression vector plasmid was constructed by subcloning a 1. 77kb E1A gene into amammalian expression vector pcDNA3 at the sites of Hind III and RamHI, and named after pcDNA3-ElA. The recombinant was cleaved with appropriate endoneuclear and sequenced. The results showed that the orientation of the insert was found to be correct, while no rearrangement was found.2. Integratuion and expression of EIA gene in Human lung adenocarcinoma cell line (Anip973)The recombinant pcDNA3-EIA was introduced Anip973 by LipofectinTM Reagent. After the cells resistant to G418, we obtained positive cell clones (Anip973-EIA). PCR, RT-PCR and immunocytochemistry assay showed that EIA gene has been integrated into Anip973-EIA cells and stably expressed.3. Effects of adenovirus type 5 EIA gene on proliferation and metastasis of human lung adenocarcinoma cells.The characteristics of the EIA expressing Anip973-EIA cells were studied in virto and viro. The expressing change of HER-2/neu gene was investigated, too. The results showed that (1) the growth rate of Anip973-EIA cells was diminished and their doubling-time increased; (2)the colony-forming efficiency of Anip973-EIA cells was inhibited significantly;(3)immunocytochemistry analysis also showed that the products of HER-2neu gene have decreased in EIA-expressing cells;(4)tumorigencity and metastasis in nude mice of Anip973-EIA were markedly suppressed. With these, we conclude that EIA gene is able to significantly suppress the malignant phenotye of human lung adenocarcinoma and the expression of HER-2/neu gene.The above results showed that EIA gene possesses important effects on gene therapy of human lung carcinoma.Our study provided data for gene therapy of tumor and possible of using EIA gene to improve tumor treatment.