[Expression and antibody preparation of FKBP38 and its application].

OBJECTIVE: To obtain recombinant N-and C-terminal of FKBP38 and prepare anti-FKBP38 polyclonal antibody for Western blotting (WB), immunohistochemical (IHC) and immunofluorescence (IF) analyses. METHODS: The N-terminal (1-207 aa) and C-terminal (209-387 aa) cDNA of FKBP38 were sub-cloned from the full-length cDNA of FKBP38 and ligated to prokaryotic expression plasmid pGEX-6P-1 for construction of the recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C. After sequencing, the recombinant vectors were transformed into E.coli BL21 and GST-tagged FKBP38-NT and FKBP38-CT were induced by IPTG. The proteins were purified by Glutathione affinity chromatography column and characterized by SDS-PAGE. Rabbits were immunized with the purified recombinant protein to prepare the antiserum, which were analyzed by WB, IHC and IF. RESULTS: The recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C were successfully constructed. After IPTG induction, the E.coli transformed with these plasmids expressed GST-tagged protein, which was successfully purified. Western blotting demonstrated that the purified antibody could specifically bind to FKBP38 in various cell lines. Immunofluorescence assay showed that FKBP38 was located mainly on the mitochondria. Immunohistochemical analysis revealed cytoplasmic location of FKBP38 in breast cells. CONCLUSION: We successfully expressed and purified N- and C-terminal of FKBP38, and FKBP38 polyclonal antibody we prepared can specifically recognize FKBP38 in SB, IF and IHC assays, which facilitates further functional investigation of FKBP38.

Down-regulation of PTEN expression due to loss of promoter activity in human hepatocellular carcinoma cell lines.

AIM: To investigate the regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene expression in human hepatocellular carcinoma (HCC) cell lines. METHODS: The mRNA and protein levels of PTEN were detected by Northern blot and Western blot in HCC cell lines, respectively. Plasmids containing different fragments of PTEN promoter with Luciferase reporter were constructed and transiently transfected into HCC cell lines to study the promoter activity. DNA analysis and RT-PCR were performed to detect the mutation of PTEN promoter and PTEN cDNA. RESULTS: Either protein or mRNA levels of PTEN in L02 cells (as a control) were significantly higher than that in HCC cell lines. The profile of PTEN promoter activity in 8 cell lines was closely correlated with levels of PTEN mRNA and PTEN protein. Furthermore, the sequence analysis of 8 cells lines showed no mutation in the region of PTEN promoter and PTEN cDNA. CONCLUSION: PTEN expression is down-regulated in HCC cell lines probably due to loss of activity of PTEN promoter.

Integrin beta1 subunit overexpressed in the SMMC-7721 cells regulates the promoter activity of p21(CIP1) and enhances its transcription.

Evidence has been emerging to suggest that integrin could induce growth inhibition in some cell types. Some of the molecular mechanisms underlying growth arrest have been elucidated. We reported here that overexpression of integrin beta1 imposed a growth inhibitory effect on the hepatocellular carcinoma cell line SMMC-7721, and this phenomenon was mainly attributed to the cyclin-dependent kinase inhibitor p21(CIP1). Furthermore, our findings suggested that transcription activity of the p21(CIP1) gene could be upregulated in the integrin beta1-overexpressing cells, and possibly controlled by the cis-elements in the core region of the p21(CIP1) promoter.

TGF-beta1-promoted epithelial-to-mesenchymal transformation and cell adhesion contribute to TGF-beta1-enhanced cell migration in SMMC-7721 cells.

Transforming growth factor-b1 (TGF-beta1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-beta1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-beta1 loses its growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-beta1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-beta1 lost its tumor-suppressive effects, but significantly stimulated cell migration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-beta1 enhanced the expression of alpha5beta1 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin (Fn). Furthermore, we observed that TGF-beta1 could also promote SMMC-7721 cells adhesion onto laminin (Ln). Our data also provided evidences that TGF-beta1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-beta1 treatment. Furthermore, TGF-beta1 induced the down-regulation of E-cadherin and the nuclear translocation of beta-catenin. These results indicated that TGF-beta1-promoted cell adhesion and TGF-beta1-induced epithelial-to-mesenchymal transformation might be both responsible for TGF-beta1-enhanced cell migration.

PKB phosphorylation and survivin expression are cooperatively regulated by disruption of microfilament cytoskeleton.

Changes in cell shape can lead to detachment and cell death, and the disruption in the actin cytoskeletal network, as one marker of cell shape changes, can itself induce apoptosis. In this study, the effects of cytochalasin B on the apoptosis-related proteins, protein kinase B and survivin were investigated. Apoptosis induced by disruption of microfilaments with cytochalasin B was found, although it happened at a low level, to simultaneously occur with G2/M arrest in 50% of the cytochalasin B-treated cells. During apoptosis, PKB phosphorylation and survivin expression were decreased by cytochalasin B, and the decline in survivin expression was preceded by PKB dephosphorylation, which implicated that survivin may be a target of PKB protein. The G2/M arrest of cytochalasin B-treated cells may be the direct function of cytochalasin B to microfilaments or the subsequent inhibition of survivin expression, or both. These results suggest that PKB/survivin signaling pathway may be responsible for the apoptosis induced by the disruption of actin cytoskeleton.

Transforming growth factor-beta1 stimulated protein kinase B serine-473 and focal adhesion kinase tyrosine phosphorylation dependent on cell adhesion in human hepatocellular carcinoma SMMC-7721 cells.

Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor and apoptosis inducer for most normal cells. However, tumor cells are commonly nevertheless sensitive to the tumor-suppressing effects of TGF-beta1. In this paper, we focus on the effects of TGF-beta1 on two important anti-apoptotic protein kinases, protein kinase B (PKB), and focal adhesion kinase (FAK), in SMMC-7721 cells. We found that PKB-Ser-473 phosphorylation was apparently up-regulated by TGF-beta1. In the meantime, PKB-Thr-308 phosphorylation was slightly up-regulated by TGF-beta1. TGF-beta1 could also enhance FAK-Tyr phosphorylation. We observed that integrin-linked kinase (ILK) was also up-regulated by TGF-beta1 in good accordance with PKB-Ser-473 phosphorylation. We first found that TGF-beta1 could stimulate PKB-Ser-473 phosphorylation possibly via up-regulating ILK expression. Furthermore, we also failed to detect PKB-Ser-473 and FAK-Tyr phosphorylation with various concentrations of TGF-beta1 treatment when cells were kept in suspension. The above results indicate that PKB-Ser-473 and FAK-Tyr phosphorylation stimulated by TGF-beta1 are both dependent on cell adhesion.