| For a long time, numerous study data indicate that RhoA protein mainly distributes in cytoplasm and on membrane, especially in cytoplasm and there is no report about the distribution of RhoA in nucleus and the mechanism of its cytoplasm-nucleus translocation. Recently, however, our previous study indicated that the distribution of RhoA in SGC-7901 cells was not only in the membrane and cytoplasm but also in nucleus. To study this phenomenon further, experiments were designed to detect the nuclear localization of RhoA in different cancer tissues and para-cancer tissue, cancer cell lines and transformed cell lines, and to study the relation between its distribution and the malignancy of the cells. Furthermore, experiments were also designed to illustrate the influencing factors of RhoA distribution in nucleus and investigate the mechanism of RhoA into nucleus. The result will be helpful to further our knowledge on the function of RhoA, the mechanisms of the migration and the invasive growth of cancer cells, and may potentially lead to novel diagnosis and therapeutic approaches.Objiective(1) To observe the localization of RhoA in human cancer tissues and para-cancer tissue, cancer cell lines and transformed cell lines, to analyse if the nuclear distribution of RhoA is related to the malignancy of the cells or not.(2) To definitude whether the distribution of RhoA in nucleus and is concerned to its gene mutation.(3) Through observing the distribution of RhoA in nucleus in human gastric cancer cell line SGC-7901 under the influence of several factors, to illustrate the influencing factors of RhoA distribution in nucleus, and discuss the mechanism of RhoA into nucleus.MethodsThe first part (1) Immunofluorescence microscopy, immunocytochemistry and Western blot assay were applied for detecting the localization of endogenous RhoA in human Gastric Cancer Cell Line SGC-7901.(2) Human gastric cancer cell line SGC-7901 cells were transfected with plasmid DNA encoding different structural RhoA (wt,63L and N19), and Western blot assay was applied for detecting exogenous RhoA in different subcellular fractions (membrane, cytosol and nucleus fractions).(3) Immunofluorescence microscopy was applied for detecting the distribution of RhoA in four cancer cell lines, including human gastric cancer cell line SGC-7901, cervix cancer cell line Hela, lung cancer cell line A549 and colon cancer cell line SW480.(4) Immunofluorescence microscopy and Western blot assay was applied for detecting the distribution of RhoA in human gastric cancer cell line SGC-7901 and transformed gastric epithelial cell line GES.(5) Immunohistochemistry staining was applied for detecting the distribution of RhoA in cancer tissues (including colon cancer, breast cancer, esophagus cancer and gastric caner), para-cancer tissues and necrotizing cancer tissues.The second partTo elucidate if the mutation of RhoA gene is related to its distribution or not by amplyfing gene DNA and testing and analysing the sequences in term of RT-PCR assay and sequencing assay.The third part(1) To observe the effect of RhoA activity on its distribution within nucleus, Western blot assay was applied for detecting the translocation of RhoA in human gastric cancer cell line SGC-7901 induced by LPA and CPT-cAMP respectively.(2) To observe the effect of RhoA modification on its distribution within nucleus, immunofluorescence microscopy was applied for investigating the translocation of RhoA in human gastric cancer cell line SGC-7901 induced by inhibiting modification of prenylation and methylation respectively.(3) To observe the distribution of RhoA within nucleus in apoptosis cells, immunofluorescence microscopy was applied for investigating the translocation of RhoA in human gastric cancer cell line SGC-7901 treated with Dexamethasone.(4) To observe the distribution of RhoA within nucleus in damaged cells, immunofluorescence microscopy was applied for investigating the translocation of RhoA in human gastric cancer cell line SGC-7901 treated with H2O2.(5) Immunofluorescence microscopy was applied to observe the effect of Taxol on RhoA distribution within nucleus through.(6) To observe the effect of LPS on the distribution of RhoA within nucleus, and to observe the role of P38MAPK in the translocation of RhoA into nucleus triggered by LPS, immunofluorescence microscopy and Western blot assay were applied for investigating the translocation of RhoA in human gastric cancer cell line SGC-7901 treated with LPS and/or P38MAPK inhibitor SB203580.(7) To observe the effect of nucleus-export inhibitor on the distribution of RhoA within nucleus, immunofluorescence microscopy was applied for investigating the translocation of RhoA in human gastric cancer cell line SGC-7901 treated with Leptomycin B (LMB).ResultsThe first part(1) In human gastric cancer cell line SGC-7901, the endogenous RhoA was localized on membrane, in cytosol and within nucleus.(2) The subcellular localization of exogenous RhoA in human Gastric Cancer Cell Line SGC-7901 includes membrane, cytosol and nucleus.(3) In cell lines including SGC-7901, HeLa, A549 and SW480, RhoA was localized not only on the membrane and in the cytosol, but also in the nucleus.(4) In human gastric cancer cell line SGC-7901 and transformed gastric epithelial cell line GES, RhoA was also found to be localized on the membrane, in the cytosol and the nucleus, but the nuclear location in SGC-7901 was more than that of GES.(5) In cancer tissues (including colon cancer, breast cancer, esophagus cancer and gastric caner), the nuclear RhoA was obviously more than that of para-cancer tissue; in the cancer tissues undergoing inflammation and necrosis, nuclear RhoA was even higher, indicating that RhoA was generally distributed in the nucleus of cells and the distribution increased when the cells undergo tumorigenesis.The second partIn human gastric cancer cell line SGC-7901, no mutation of RhoA gene was detected.The third part(1) In human gastric cancer cell line SGC-7901, LPA induced membrane and nucleus translocation of RhoA and CPT-cAMP induced cytosol translocation of RhoA.(2) Neither the inhibition of prenylation nor the inhibition of methylation had any significant effect on the distribution of RhoA within nucleus in human gastric cancer cell line SGC-7901, which suggested that the cytoplasm-nucleus transference of RhoA was not affected by the modifications of RhoA protein.(3) In apoptotic cells, the distrubution of RhoA within nucleus was reduced and RhoA was transfered to membrane.(4) In damaged cells, the amount of RhoA increased, and more RhoA was translocalized into the nucleus.(5) Toxal could trigger the translocation of RhoA from nucleus towards cytosol and membrane, and LPA could enhance this translocation, which suggested that the distribution of RhoA was related to the microtubule structure in the cells.(6) In the cells undergoing inflammation, the nuclear RhoA increased significantly, the translocation of RhoA from cytosol toward nucleus was obvious, some cells even showed no RhoA distribution in cytosol, and the nucleus moved largely towards one side of the cell. The P38MAPK inhibitor SB203580 could effectively hinder the LPS- triggered translocation of RhoA into nucleus at initial stages.(7) The LMB had no significant effect on RhoA distribution within nucleus.Conclusion(1) Nuclear localization of RhoA is all-pervading, and its nuclear distribution is related to the malignancy of the cells.(2) The distribution of RhoA in cancer cell was not related to its gene mutation. (3) Illustrating the influencing factors of RhoA distribution in nucleus:?The activation of RhoA can increase its translocation from cytosol towards nucleus and membrane and inhibition of its activity had opposite effect.?In apoptotic cells, the distrubution of RhoA within nucleus reduced and transfered to membrane.?In damaged and inflammatory cells, the amount of RhoA increased, and more RhoA was translocalized into nucleus.?Taxol can hinder translocation of RhoA from cytosol towards nucleus.?The protein modification inhibitor and nucleus-export inhibitor had no effect on the distribution of RhoA in nucleus, suggesting that the transport of RhoA into nucleus is diffusive process and not affected by the modification of the protein. |
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