Effects Of LyGDI And Its Mutant On The Growth, Metastasis And Radiation-sensitivity Of L929 Cells

Objective:LyGDI(27kDa),also named RhoGDI2,D4-GDI or RhoGDIβ,a major member of Rho GDP dissociation inhibitor(RhoGDI) family,has been implicated in the regulation of many aspects of cell motility and invasion,including cell polarity, cytoskeletal organization,and transduction of signals from the extracellular environment. This study is to investigate effects and mechanisms of LyGDI and its mutant on the growth,metastasis and radiation-sensitivity of the L929 cell line,which is tumorigenic in nude mice.Methods:(1)The LyGDI and D19LyGDI genes,which were constructed with the pEGFP vectors,and the empty vector pEGFP-C1 were transfected into L929 cells by using Lipofectamine^TM 2000 respectively.To obtain stable cell lines,cells were incubated in RPMI 1640 medium containing G418 for about 3 weeks.Then,the expression of EGFP was detected with confocal microscopy;the expression of fusion proteins was detected by Western Blot analysis.(2)The MTT assay was performed to detect the effect of LyGDI and D19LyGDI on the growth of L929 cells.(3)In vitro wound-closure assay was performed to observe effects on the invasion and metastasis of L929 cells.(4)The colony formation testing was performed to determine whether these two genes enhance the radiation-sensitivity of L929 cells.(5)Flow cytometry was utilized to quantify the cell cycle distribution after cells were irradiated by X-ray,in order to detect the effect of exogenously introduced genes combined with irradiation on cell cycle distribution.Results:(1)The green fluorescence of EGFP was detected with confocal microscopy in all transfectants.(2)Western Blot analysis revealed that there were corresponding fusion proteins in all transfectants.(3)The MTT assay showed an obviously slower growth in transfectants,especially in pEGFP-D19LyGDI-L929 cells.(4) The wound-closure assay showed decrease of the mobility in transfectants,also especially in pEGFP-D19LyGDI-L929 cells.(5) Dose-survival curve derived from the colony formation testing showed that, radiation-sensitivity parameters(D_o,D_q and SF₂)of transfectants were lower than those of L929 and pEGFP-C1-L929 cells,and the least of pEGFP-D19LyGDI-L929 cells.(6)All cells irradiated by 4 Gy X-ray showed that,the ratio of G₁ was increased,and this change of pEGFP-LyGDI-L929 cells was more obvious than others.Conclusion:The stable cell lines were established;the introduction of LyGDI and D19LyGDI inhibitted the growth,the ability of invasion and metastasis,and enhanced the radiation-sensitivity of L929 cells,which were more obvious in pEGFP-D19LyGDI-L929 cells;exogenously introduced LyGDI promoted the radiation-induced G₁ arrest,thus,the radiation-sensitivity of L929 cells was enhanced.