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Objective: our study was to investigate the effect of hepaCAM gene on cell cycle of bladder cancer cell line T24 and BIU-87. Method: Adenovirus vector with hepaCAM was infected into T24 and BIU-87 cells. The expression of hepaCAM mRNA and protein were measured byRT-QPCR and Western-blot. Immunofluorescence detected the cellular localization of hepaCAM. The distribution of cell cycle was analyzed by FCM. The protein expression of cyclinD1 was measured by Westein-blot. Result: The expression of hepaCAM mRNA and protein was increased in two cell lines infected with pAdH5-hepaCAM. The G0/G1 cycle was increased in infected cells, and the protein expression of cyclinD1 was decreased in same cells. Immunofluorescence revealed that hepaCAM protein localized on cytoplasm in well-spread cells, otherwise it localized on the junction of each cell. Conclusion: T24 and BIU-87 cells that infected pAdH5- hepaCAM was blocked in G0/G1 cycle, and it can decreased the protein expression of cyclinD1.
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Objective: To investigate the effects of extracellular domain-truncated mutant of hepaCAM gene on biological behaviors of bladder carcinoma cells in vitro. Methods: Human cell line T24 was transfected with pEGFP-N2-hepaCAM, pEGFP-N2-hepa CAM-mt or mock plasmid pEGFP-N2 by lipofectamine mediation. The stable colonies were obtained by G418 screening. Laser confacal microscopy was used to detect the location of hepaCAM-mt in the T24 cells, Western blotting was used to determine the expression of protein in stable cells. Cell adhesion and motility abilities were tested by cell adhesion and detachment assays and wound healing and Matrigel invasion assays. The proliferation ability was investigated by MTT assay. Results: In well-spread cells, mutant proteins were localized on the perinuclear membrane like wild-type hepaCAM protein; after the cells contacted with each other, hepaCAM-mt protein had non-specific expression in the cells, which was significantly different with the localization of the hepaCAM proteins. T24 cell clone which acquired stable expression of hepaCAM-mt protein had decreased adhesion and motility abilities and enhanced proliferation potential compared with the cells with wild-type hepaCAM gene expression. Conclusion: The deletion of extracellular domain in hepaCAM gene was important for the localization of hepaCAM protein but had no significant effect on the proliferation, adhesion, and migration of T24 cells. The extracellular domain of hepaCAM was necessary for exerting normal functions of hepaCAM gene.
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Objective: This study was to evaluate the effects of hepaCAM (hepatocyte cell adhesion molecule) on invasiveness of human renal cell carcinoma (RCC) cell line 786-0 and explore the underlying mechanism. Methods: A recombinant plasmid containing hepaCAM cDNA (hepaCAM-pEGFPN2) was transfected into 786-0 cells, then the positive cell clones were selected with G418 and amplified. The 786-0 cells transfected with pEGFPN2 and untreated 786-0 cells were as controls. The mRNA expressions of hepaCAM and matrix metalloproteinase (MMP)-2 and MMP-9 before and after stable transfection are detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time fluorescence PCR. The activities of MMP-2 and MMP-9 were analyzed by gelatin zymography. The invasiveness of 786-0 cells was determined by Transwell migration assay in vitro. Results: The mRNA transcription of hepaCAM was significantly increased in 786-0 cells after stable transfection of hepaCAM (P <0.01). The mRNA expressions and activities of MMP-2 and MMP-9 in 786-0 cells were markedly inhibited by hepaCAM (P <0.05). The transmembrane migration capacity of cells was significantly reduced after stable transfection of hepaCAM (P <0.01). Conclusion: HepaCAM inhibits the activities of gelatinase by down-regulating the expressions of MMP-2 and MMP-9, thereby preventing the invasiveness of renal cell carcinoma 786-0 cells.
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Objective:To explore the effect of gene hepaCAM transfection on the proliferation in human bladder carcinoma cell line. Methods: Plasmid pEGFP-N2-hepaCAM was transfected into T24 cells by Lipofectamine2000, and the T24 cell stably expressing hepaCAM gene was established by G4l8 selection.The expression of hepaCAM mRNA and protein was detected by RT-PCR and Western blot assays. The effects of hepaCAM on cell proliferation were measured by MTT and colony formation assays. The cell cycle progression were measured by flow cytometry. The expression of CyclinD1 mRNA was detected by RT-PCR. Results:Expression of hepaCAM gene and protein were proved by RT-PCR and Western blot assays. The MTT assay showed cell growth in the T24/hepaCAM-GFP group was significantly slow when compared with non-transfection and mock-vehicle groups whereas the rates of clonal formation were greatly decreased(P
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Objective:To investigate the effect of transfection with wild hepaCAM gene on biological behavior of the transitional cell carcinoma of bladder in vitro.Methods:Human TCCB cell line T24 was transfected with pEGFP-N2-hepaCAM,mock plasmid pEGFP-N2 with lipofectamine.Laser confocal microscopy was used to detect the location of hepaCAM in cells.Western bolt was used to determine the expression of target of hepaCAM in stable cells.The cell adhesive and motility ability were tested by cell spreading assay and matrigel invasion assay.Cell proliferation ability was investigated by MTT assay。Results:In well spread cells,hepaCAM localized on the perinuclear membrane,whereas in the cells which contacts,hepaCAM predominantly accumulated at the sites of cell-cell contacts on cell membrane.Cell clone with stable expression of hepaCAM,was acquired.In vitro experiments as cell spreading assays and matrigel invasion assays showed the cell adhesion and cell motility properties of hepaCAM group were apparently enhanced compared with the non-transfection or mock-vehicle groups.The MTT assay showed cell proliferation ability in the hepaCAM group was notably decreased when compared with the non-transfection or mock-vehicle groups.Conclusion:The hepaCAM gene can restrain malignant phenotypes of the human TCCB cells in vitro,and may inhibit the TCCB invasion and metastasis by influencing the function of some adherence and proliferation factors.[
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Objective:hepaCAM (hepatocyte cell adhesion molecule)is a kind of immunoglobulin superfamily member which was discovered as a tumor suppressor. This study was to investigate the effects of hepaCAM transfection on proliferation and invasion of human renal cancer cell line 786-0. Methods:An eukaryotic expression plasmid containing hepaCAM,hepaCAM-pEGFPN2 was transfected into 786-0 cells,then positive cell clones were selected with G418 and amplified. pEGFPN2 transfected cells and untreated cells were set as controls.The expression of hepaCAM was detected by reverse transcription-polymerase chain reaction (RT-PCR). Proliferative ability of 786-0 cells was measured by MTT assay and the invasive ability in vitro was determined by transwell chambers method. Results: Plasmid pEGFPN2,hepaCAM-pEGFPN2 were successfully constructed and stably transfected into 786-0 cells.After transfection,the inhibitory rates of cell growth at the fourth、 fifth and sixth day were 26.5%,38.1% and 35.7%,respectively. The penetrating cells were significantly less in 786-0/ hepaCAM-pEGFPN2 group than in 786-0/pEGFPN2 and 786-0 groups (24.6?5.3vs35.5?6.2 and 37.1?7.0,P