α1,2-Fucosyltransferase Gene Transfection Influences Biological Behavior Of Ovarian Carcinoma-derived RMG-I Cells

PrefaceThe mortality of ovarian carcinoma is the highest in female genital malignancy. The pathogenic mechanism is unclear, early diagnosis is ineffective and therapeutic efficacy is bad. Conclusive mechanism of invasion and metastasis, which are responsible for poor survival rate, is important to metastasis prevention and treatment directions. Lewis y antigen is overexpressed in 70% of ovarian carcinoma. It has been shown that the structure of CA₁₂₅ includes Lewis y. So Lewis y is related to ovarian carcinoma obviously. In our studies, ovarian carcinoma cells RMG - I are transfected by α - 1, 2FT gene and cellular models, which highly express Lewis y are constructed.Cellular biological characteristics influenced by α - 1,2 FT are observed by methods of biochemistry and immunochemistry. The relationship between Lewis y and genesis, infiltration and metastasis of ovarian carcinoma is discussed.Materials and Methods一,Materials1. Cell line, plasmid and bacterial strainPlasmid pcDNA3. 1, recombinant HFUT - H containing α1 ,2 - FT gene and human ovarian carcinoma cell line RMG - I are offered by Professor Lin in Obstetrics and Genecology of Shengjing hospital.2. AgentsEndotoxin - free ultrapure plasmid DNA purification kit, cell phect trans-fection kit, G₄₁₈, mouse anti - Lewis y monoclone antibody, SABC kit, DMSO, DMEM , fetal calf serum._<sub>N Methods1. Extraction of HFUT - H and pcDNA3. 1: extracted plasmid according to the direction of cell phect transtection kit.2. Transfection and resistant cell colon screening: RMG - I were trans-fected by HFUT - H and pcDNA3. 1 with cell phect transfection kit. And screened the resistant clone with G418.3. Immunocytochemistry: the trial was carried out according to the direction of SABC kit.4. Observation of cell morphology: observed cells with invert microscope and HE dying.5. Proliferation experiment: drawn growth curve by MTT and cell counting.6. Soft agar culture;cells were inoculated in double layer agar plate, and 14 days later, counted.7. Analysis of cell cycle by flow cytometry;after PI dying, cells were detected by flow cytometry.8. Statistical analysisStatistical analysis was performed by the SPSS11.0 package. Probability of P<0.05 was considered statistically significant.Results1. Extraction of HFUT - H and pcDNA3. 12. Immunocytochemistry: the positive rate of Lewis y expressed in RMG -I - H is higher than in RMG - I and RMG - I - pcDNA3. 1, P <0. 05.3. Observation of cell morphology: RMG - I and RMG - I - pcDN A3. 1 growth in lamellar shape and the borderline is regular, but the borderline of RMG - I — H is irregular and cells are easy to shed from culture flask and grow superposably.4. Growth curve;RMG - I - H grows faster than the other two significant-ly-5. Soft agar culture;rate of clone formation;RMG - I - H is 35% , RMG - I and RMG- I -pcDNA3.1 were 13% and 12% separately, P<0.05.6. Analysis of cell cycle: compared with RMG - I and RMG - I - pcD-NA3. 1, percentage of RMG - I - H cells decreases in Gt phase and Go phase and increases in G2 phase and in M phase.DiscussionsThe glycocalyx layer of mammalian cells is known to change in association with cellular adhesion, recognize, canceration, invasion and metastasis. There are sufficient evidences that Lewis antigens are tumor - associated molecules and correlated to high grade and poor prognosis tumors.We transferred al ,2 - FT gene into RMG - I cells and constructed cellular model expressing al ,2 - FT gene and Lewis y highly firstly. The expression of Lewis y was found to increase in RMG - I - H by immunocytochemistry, biological behavior of which changed.The reasons of that maybe: ( 1 ) modifications of glycosyltransferase make tumor cells escape from immunity;(2) negative charge increases and cellular repelling force increases, which make cells easy to fall from tumor;( 3 ) LS path is an important way of metastasis;(4 ) Lewis y glycolipid plays an important role in the mechanism of cancer metastasis via regulating the procoagulant activity of CCA — 1;( 5 ) Lewis y alters the structure of proliferation factor receptor on cellular membrane.The high frequency of Lewis y -expressing tumors, its high density and altered expression on the surface of tumor cells, and its relatively homogenous expression in primary and metastatic lesions have led to its selection as an antigenic target for a range of epithelial tumors. Some trials in abroad have already been in clinical phase 1. Lewis y recognizing antibody and vaccine are desirable therapy of ovarian carcinoma.Conclusions1. Expression al ,2 - FT gene leads to high expression of Lewis y in RMG - I cells.2. Lewis y and al ,2 - FT enhances proliferation and growth rate of RMG -I cells and encourage tumor growing.3. Lewis y is one of reasons which are responsible for genesis and progress of ovarian carcinoma.4. Lewis y antibodies and vaccines maybe desirable treatment of ovarian carcinoma.