| INTRODUCTION: Ndr2 is a newly found member of Ndr gene family which consists of Ndrl/RTP/Drgl, Ndr2, Ndr3 and Ndr4. The precise molecular and cellular function of members of this family is still unknown. But they are known to be involved in cellular differentiation events. Ndrl was first identified, and present investigation suggests it might be related to differentiation and anti-oncogenesis. Human Ndr2 was found by subtractive hybridization of human glioma tissue with its normal counterpart. Our preliminary investigation showed that the mRNA expression patterns of Ndr2 is dominantly in salivary gland, brain, skeletal muscle and mammary gland, and low expression in bone marrow, testis, periperal blood, and placenta, but it did not expressed in leukocyte, colorectal and some tumor cell line such as, Hela S3, leukemia cell, Bukitt's lymphoma, adenocarcinoma SW480. The distinct expression patterns of the four family members suggest that Ndr2 might have relationship withtumor.AIM: To explore whether Ndr2 is related to tumor development, the expression of Ndr2 in lung cancer tissue and its role in lung cancer cell were observed.METHODS: RT-PCR analysis of Ndr2 expression in lung cacinoma specimens was used. Lung carcinoma specimens were taken from liquid azote and homogenized soaked in Trizol, Total RNA was isolated and quantified. Total RNA was used for RT-PCR reaction to amplify Ndr2. Western blotting assay was used . The frozen tissue was homogenized soaked in lysis buffer, the total protein quantity was determined by quantitative kit, and then applied to SDS-PAGE. The separated protein on SDS-PAGE was electro-transfered onto NC membrane. Western blotting assay was carried out as routine method described as reference . The routine immunohistochemistry was carried out using anti-Ndr2 monoantibody. Stable transfection and inducible expression of Ndr2 were used in lung adenocarcinoma ceh1. Full length cDNA encoding human Ndr2 was inserted into ecdysone-inducible vector pIND, named as pIND-Ndr2. Recombinant vector containg Ndr2 and another helper vector pV were co-transfected into GLC-82 lung carcinoma cells. Single clone resistant to both zeocin and G418 was isolated and cultured for enlarging its number. Cells were induced by ponasterone A , and the expression of Ndr2 was assayed by RT-PCR and Western blotting as described above. Cell growth is assayed by MTT method. Row Cytometry was used to determine cell cycle. To observe the expression of Ndr2, recombinant vector adenovirus-p53 was transfected in A549 lung carcinoma cell.RESULTS AND CONCLUSIONS: To explore whether Ndr2 is related to tumor development, its expression was evaluated firstly in 51 cases lung-carcinoma and equivalent number of normal tissue collected from clinical operation. RT-PCRassay indicated that according to tumor-free lung tissue, displayed completely negative in 9 of 26 cases of squamous carcinoma , low expression in 8 of 26 cases of squamous carcinoma; displayed completely negative in 4 of 14 cases adenocarcinoma, low expression in 3 of 14 cases adenocarcinoma; and displayed completely negative in 4 of 11 cases small cell lung carcinoma, low expression in 4 of 1 leases small cell lung carcinoma. RT-PCR results were confirmed by Western blotting and imrnunohistochernistry by using monoclonal antibody to Ndr2. The data above demonstrated that Ndr2 displayed down-regulated expression in lung carcinoma tissue in both transcription and protein level. These data indicated that Ndr2 expression was down-regulated in lung carcinoma, and related to tumor grade and phase development. The lower the tumor grade is, the higher the negative rate of Ndr2 expression is. The later the tumor phase is, the higher the negative rate of Ndr2 expression is. Additionally, human lung carcinoma cell line GLC-82 stably transfected with Ndr2 was acquired and the induced over-expression of Ndr2 resulted into Gl phase inhibition, and the growth of the tumor cell was reduced, and the clone forming ability was also inhibited, the expression of cy...
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