Overexpression Of Annexin I In Malignant Transformed BEP2D Cells By α-Particle And Lung Tumorous Tissues And Effect Of Annexin I Gene Transfection On The Growth Of BEP2D Cells

Lung cancer is one of the major causes of cancer deaths in the would. Epidemiological studies have shown that a - particle emitted by radon and its progeny products is one of the most important factors for inducing lung cancer. Domestically, exposure to radon represents the biggest involuntary environmental hazard. About 50% average annual effective dose of natural irradiation resulted from a - particles emitted by radon and its progeny products. The mechanism of lung cancer induced by a — particles emitted by radon and its progeny products was more and more concerned.Early research in our laboratory showed that Annexin I gene was one of the genes upregulated in malignant transformed rat bronchial epithelial cells, indicating that the change of Annexin I expression might involved in the transformation process. Recently, more and more researches showed the relationship between the upregulation of Annexin I expression and the development of various cancers. But till now there is no data available about the relationship between the change of Annexin I expression and lung cancer.In order to explore the relationship between the expression of Annexin I, a calcium - dependent phospholipid binding protein, and lung cancer, the role of increased Annexin I gene expression in tumor and cellular malignant transformation process, an immortalized human bronchial epithelial cell line BEP2D, which was established by transfecting human papillomavirus(HPV18), was used as a model. This cell line hasobvious advantages in the functional study of Annexin I in the transformation process compared with the reported models by tumor cell lines.In this study the expression level of Annexin I in both the immortalized and malignant transformed BEP2D cells by a- particles emitted by Pu238 (3.94Mev) had been determined by western blot and northern blot. The distribution of Annexin I was also studied by cellular immunochemistry. Moreover, the expression change of Annexin I in nontumorous and tumorous lung tissues from 4 lung adenocarcinoma and 8 squamous cell carcinoma patients were observed by western blot. Furthermore, the mammalian expression vector of Annexin I (pCI - AXI) was constructed by digesting the Annexin I cDNA containing plasmid pT7T3D (No.557597 from the American Type Culture Collection) by Not I and Xho I, directional subcloning into mammalian expression vector pCI - neo containing the CMV promoter (Promega Co.), and was transfected into the immortalized human bronchial epithelial BEP2D cells using UPOFECTAMENE (GIB-CO) to oberseve the effect of Annexin I on the growth of BEP2D cells. The anti - sense oligonucleotide was also synthesized and tested. The following results were observed:1. Northern blot and Western blot showed that the expression level of Annexin I was moderate in immortalized BEP2D cells, but was overexpressed in a - particles transformed BEP2D cells. The amount of expression is 1.5 times higher in transformed BEP2D cells than that in the immortalized ones both in mRNA and protein levels.2. In BEP2D cell Annexin I distributes on membrane and in plasma, but not in nucleus. However no difference of distribution between immortalized BEP2D cells and a - particle transformed BEP2D cells were observed.3. More Annexin I in all the 4 lung adenocarcinoma and 8 squamous cell carcinoma patients was expressed in the lung tumor tissues than in the lung nontumorous tissues, which derived from the same case. The optical density analysis showed that the ratio of expression in the tumorous tissues compared with the nontumorous tissues is 2.7 (p<0.01).4. Compared with BEP2D cells and pCI - neo transfected cells, the pCI - AXI transfected cells was 10 hours and 6 hours less of the Population Double Time (PDT)respectively, more cells went into S phase and G1/M phase by flow cytometric analysis. The anti - sense oligonucleotide significantly suppressed the growth of BEP2D cells at 50 nM (p<0.05).5. The pCI - AXI transfected cells also showed increased clonogenicity in plating efficiency (p<0.002) and soft agarose assays, and loss of density inhibition growth.This research first reports that the overexpress of Annexin I is related to lung cancer and BEP2D cell malignant transformation, and may be a factor implicated in lung tumorigenesis. These results also indicate that upregulated expression level of Annexin I can promote the growth and survival ability of immortalized BEP2D cells. It is suggested that these properties of Annexin I may be helpful for the malignant transformation of cells and the development of cancers, specially at early stage, combined with reported results. The significance of the Annexin I overexpression in cell transformation and cancers may provide a new indicator and a new target for early diagnosis and treatment of cancers.