S-phase delay in human hepatocellular carcinoma cells induced by overexpression of integrin β1

Figure 1 Integrin α5 and β1 protein levels in α5-, β1- and α5β1 transfected SMMC-7721 cells. (A) The expression level of α5 chain was increased in α5-7721 and α5β1-7721 cells. (B) Two forms of β1 integrin were due to different levels of β1-chain glycosylation. The hypoglycosylated lower band was tenta-tively identified as biosynthetic precursor of β1 subunit, the hyperglycosylated upper band was mature subunits, exposed in part on the cell surface (the 130-kDa product). Immunoblot assay showed that protein levels of the mature form were el-evated in the transfectants, especially in β1-7721 and α5β1-7721 cells. (C) The protein level of β-actin showed an equal loading amount in each well. Lane 1, mock cells; lane 2, α5-7721; lane 3, β1-7721; lane 4, α5β1-7721. Each point in the graphs was the mean value from three separate experiments.

Figure 2 S-phase delay was induced in β1- and α5β1-transfectant cells. For cell cycle analysis, transfected and mocked cells were synchronized by exposure to SFM for 48 h, then grown in RPMI1640 medium containing 10% CBS and penicillin/streptomycin solution. Twelve or 16 h later, the cells were collected and analysed for flow cytometry as described under “Materials and Methods”. A, mocked cells; B, α5-7721 cells; C, β1-7721 cells; D, α5β1-7721 cells. Each bar in graph represented the mean ± SD obtained from three independent experiments. The S-phase delay was significantly different in β1-7721 and α5β1-7721 cells (n = 3, ^bP < 0.01 vs mocked cells).

Figure 3 Message RNA and/or protein levels of cell cycle regu-latory genes p21^cip1 and p27 ^kip1 in β1-7721 and α5β1-7721 transfectants, but not that of cyclin D1 and cdk2. (A) Immunoblot assay showed the protein level of cyclin D1 and cdk2 were not apparently affected, but p21^cip1 and p27^kip1 pro-tein levels were increased in β1-7721 and α5β1-7721 cells. The protein level of β-actin was detected to assess the loading amount in each well in SDS-PAGE gel. (B) Message RNA lev-els of p21^cip1 and p27^kip1 were assessed by RT-PCR, and nor-malized by that of β-actin. It was apparent that mRNA level of p21^cip1 was increased in β1- and α5β1- transfected cells. However, the p27^kip1 mRNA amount was the same as control. Each result represented three separate experiments. Lane 1, mocked cell; lane 2, α5-7721; lane 3, β1-7721; lane 4, α5β1-7721.

Figure 4 Activation of PKB, but not FAK, was downregulated in β1- and α5β1- transfected cells. (A) FAK activation was as-sessed by phosphotyrosine- specific antibody, followed by stripping and reprobing with anti-FAK antibody. (B) PKB and its Ser473-phosphorylated forms were determined by 10% SDS-PAGE with the equal loading amount. The loading amount control is shown in Figure 1C and Figure 3A. Lane 1, mock cells; lane 2, α5-7721; lane 3, β1-7721; lane 4, α5β1-7721. Results were representative of 4 repeated experiments.

Figure 5 Ser473-phosphorylated form of PKB was decreased, but p27^kip1 protein level was increased concomitantly in SMMC-7721 parental cells treated with the PI3K inhibitor wortmannin. The parental SMMC-7721 cells were starved with serum-free medium for 48 h, then grown in normal medium/10% CBS containing DMSO (as control, 24 h) or 100 nM wortmannin for the indicated times. The amount of DMSO did not exceed 0.1%, which was determined not to damage the cells. Equal amount of wortmannin was added again after grown for 24 h. The level of Ser473-phosphorylated form of PKB (arrow) was declined, but p27^kip1 protein level was elevated with the treatment of wortmannin in SMMC-7721 cells. Results were representative of at least 3 repeated experiments. Abbreviation: DMSO, dimethylsulfoxide; WT, wortmannin.

Figure 6 β1-7721 and α5β1-7721 cells subjected to spreading on fibronectin- or laminin- coated culture dishes. The mocked and transfected cells were plated on FN- or LN- coated tissue culture plates in normal medium for 60 min, then cells on the plates were photographed by phase-contrast microscopy with a digital camera. Abbreviations: FN, fibronectin; LN, laminin.

Figure 7 Effects of laminin on transfected cells. Protein levels of phosphorylated FAK (A) and Ser473-phosphorylated form of PKB (B) were increased in the transfected cells, especially in β1-7721 and α5β1-7721 cells grown in the LN-coated culture dishes for 60 minutes. (C) Under the same condition, p27^kip1 protein level was decreased in β1-7721 and α5β1-7721 cells. Lane 1, mock cells; lane 2, α5-7721; lane 3, β1-7721; lane 4, α5β1-7721. Each result represented at least 3 independent experiments.

Figure 8 The percentage of cells in S phase was increased in SMMC-7721 cells plated on poly-HEME-coated petri dishes. The parental SMMC-7721 cells were plated on poly-HEME-coated petri dishes, and cultured in normal medium for 24 h (A), 48 h (B) or 72 h (C), respectively, then collected and analysed by flow cytometry. The cell cycle pattern (C) was simi-lar to that of β1-7721 or α5β1-7721 cells.