Focal adhesion kinases crucially regulate TGFß-induced migration and invasion of bladder cancer cells via Src kinase and E-cadherin.

Focal adhesion kinase (FAK) is a non-receptor protein-tyrosine kinase that is triggered off by special extracellular signals such as some growth factors and integrins. FAK is found in cell-matrix attachment sites and implicated in cell migration, invasion, movement, gene expression, survival and apoptosis. In this study, we aimed to investigate whether FAK plays a role in invasion and migration of bladder cancer cells. Using an FAK-specific small interfering RNA (siRNA) and an FAK inhibitor PF-228, we found that inhibition of FAK tyrosine phosphorylation or knockdown of FAK suppressed invasion and migration of bladder cancer cells. Src is an important mediator of FAK-regulated migratory and invasive activity. Tyrosine phosphorylation of Src and FAK is mutually dependent and plays a key role in transforming growth factor beta (TGFß)-induced invasion and migration. E-cadherin acts downstream of FAK and is a critical negative regulator in FAK-regulated invasion and migration of bladder cancer cells. These findings imply that FAK is involved in oncogenic signaling of invasion and migration, which can be a novel therapeutic target to treat patients with bladder cancer.

Monitoring of prostate cancer growth and metastasis using a PSA luciferase report plasmid in a mouse model.

OBJECTIVE: To construct a PSA luciferase report plasmid and monitor the growth and metastasis of prostate cancer after emasculation in SCID mice. METHODS: PSA promoter sequence and luciferase gene were amplified by PCR and subsequently inserted into pZsGreen1-1 vector to construct pPSA-FL-Luc vector. LNCaP cells that were stably transfected with pPSA-FL-Luc were used to establish a SCID mouse xenograft model. Then, the growth and metastasis of prostate cancer were monitored via living imaging. RESULTS: We successfully constructed a PSA luciferase plasmid, pPSA-FL-Luc. DHT enhanced luciferase activity in a concentration-dependent manner in 293T cells with pPSA-FL-Luc transfection. Prostate cancer SCID mouse model was established with pPSA-FL-Luc transfected LNCaP cells. In tumor bearing mice with or without emasculation, pPSA-FL-Luc plasmid was applied to monitored tumor growth and metastasis based on bioluminescence imaging. CONCLUSIONS: We construct a pPSA-FL-Luc plasmid, which stably expresses luciferase and can be applied to monitor tumor development in a prostate SCID mouse model.

Ureteral obstruction and urinary fistula due to fibrin glue after partial nephrectomy: A case report and review of the literature.

In the present study, we report the case of a 69-year-old female who developed urinary leakage following partial nephrectomy (PN) to remove left renal masses. The results of CT and MR urography revealed left proximal ureteral obstruction and urinary fistula. Reoperation was performed on the 16th postoperative day to explore the left kidney and ureter in order to relieve the obstruction. The left proximal ureter was found to be enfolded by fibrin glue and showed marked stiffness and adhesion during the reoperation. The lesion of the ureter was resected and the ureter was anastomosed with the routine double-J stent. Pathological examination of surgical specimens revealed fat fibrous scar tissue hyperplasia with inflammatory cell infiltration. The patient recovered completely without exudate. Our experience suggests that care should be taken to avoid touching the ureter with fibrin glue during PN surgery.

[Construction of oxalate-degrading intestinal stem cell population in mice].

OBJECTIVE: The oxalate-degradation genes of Oxalobacter formigenes (Ox.F)-frc gene and oxc gene-were cloned and transfected into intestinal stem cell population of the mouse to make the latter obtain oxalate-degradation function. METHODS: (1) The dicistronic eukaryotic expression vector, which could express oxc gene and frc gene in the same time, pIRES-oxc-frc was constructed. (2) The intestinal stem cell population of the mouse were isolated and cultured, and the function of the cell growth and differentiation was identified. (3) The cells were transfected with pIRES-oxc-frc. After selection by G418, the function of the cell growth and differentiation and the the expression of the objective genes were identified. (4) The concentration of the oxalate in the culture medium which was used to culture the transgenic cells was determined by ion chromatography to explore the oxalate-degradation function of the cells. RESULTS: Ox.F could be isolated and cultured from the feces of Chinese people. Compared with the foreign reports, a certain morphologic variation of the Ox.F existed. But the oxc gene and frc gene showed high homology with the sequence reported in GenBank. The recombinant plasmid containing oxc gene and frc gene could successfully be transfected into the intestinal stem cell population of the mouse. The expression of the objective genes was normal. The concentration of the oxalate in the culture fluid of the transgenic intestinal stem cell population [(2.48 +/- 0.03 g/L)] was lower than those of the normal group [(2.69 +/- 0.01) g/L] and the control group [(2.69 +/- 0.01) g/L, P < 0.01]. CONCLUSION: The oxc gene and the frc gene could be transfected into the intestinal stem cell population of the mouse, and the cells could be given oxalate-degrading function. The gene of prokaryocyte could be introduced into the eukaryocyte for a successful expression.

Estrogen in combination with 5-azacitidine up-regulates p75NTR expression and induces apoptosis in 22Rv1 prostate cancer cells.

Previous studies suggest that the low-affinity neurotrophin receptor p75NTR inhibits the proliferation of human prostate cancer cells, and that estrogen interacts with p75NTR in many tissues. In this study, we exposed 22Rv1 androgen-independent prostate cancer cells to 17-ß-estradiol and the DNA demethylating agent 5-azacitidine (5-AzaC) to explore the interactions between estrogen and p75NTR. We found that estrogen induced estrogen receptor (ESR) subtype?2 and p75NTR expression in 22Rv1 cells, and that 5-AzaC further enhanced these effects. Estrogen in combination with 5-AzaC induced cell apoptosis, which was associated with the inhibition of NF-κB translocation to the nucleus. These results provide evidence that the up-regulation of ESR2 and p75NTR by estrogen plus 5-AzaC may be a potential therapeutic strategy for the treatment of androgen-refractory prostate cancer.

Stable expression of the oxc and frc genes from Oxalobacter formigenes in human embryo kidney 293 cells: implications for gene therapy of hyperoxaluria.

Hyperoxaluria can lead to multiple pathologic conditions such as recurrent urolithiasis, oxalosis, nephrocalcinosis and even renal failure, but there is no known oxalate-degrading pathway in the human body, and current therapeutic options for patients with hyperoxaluria are limited. Oxalyl-CoA decarboxylase and formyl-CoA transferase are the key enzymes in the oxalate catabolism of Oxalobacter formigenes which dwell in the intestine of vertebrates and have an important symbiotic relationship with their hosts. The aim of this study was to insert the oxalate-degrading enzyme genes into human embryo kidney (HEK) 293 cells and to evaluate if the oxalate-degrading enzymes could be expressed in these cells and keep their enzyme activity. We present here the cloning of the two genes from O. formigenes and their subsequent expression in HEK293 cells. The results showed that the expression of oxalyl-CoA decarboxylase and formyl-CoA transferase was confirmed by RT-PCR and Western blotting, and the proteins were located in the cytoplasm of transfected cells. Furthermore, the transfected cells were capable to a certain degree of degrading oxalate. These findings suggest that the transfer of oxalate-degrading enzyme genes into human cells is possibly a potential candidate for the gene therapy of hyperoxaluria.

[Antisense targeting to human papillomavirus 18 E6/E7 affects the proliferation and apoptosis of human cervical carcinoma: an in vitro experiment with HeLa cells].

OBJECTIVE: To investigate the effect of eukaryotic fluorescent expression vector carrying antisense human papillomavirus (HPV) 18 E6/E7 on the growth and proliferation of human cervical carcinoma. METHODS: The HPV18 E6E7 with the length of 716 bp was amplified by PCR, the PCR product was inversely inserted into the eukaryotic fluorescent expression vector pEGFP-C1 so as to construct the recombinant eukaryotic expression plasmid pEGFP-HPV18E6E7as (EGFP-18AS). Human cervical carcinoma cells of the line HeLa were cultured and randomly divided into 3 groups: Group A transfected with the recombinant plasmid EGFP-18AS, Group B transfected with the blank plasmid pEGFP-C1, and Group C without transfection used as control group. The mRNA expression of HPV 18 E6/E7 in the HeLa cells was detected by RT-PCR and protein expression of HPV18 E6/E7 HPV 18 E6/E7 in the HeLa cells was detected by. Western blotting MTT assay was performed to dynamically monitor the surviving cells and the cell apoptosis was observed by flow cytometry and fluorescence microscopy. RESULTS: The protein and mRNA expression levels of HPV18 E6/E7 in the HeLa cells transfected with HeLa/18AS were both remarkably lower than those in the HeLa cells transfected with blank plasmid and those of the control group. The numbers of surviving HeLa cells of Group A was significantly lower than those of Groups B and C (both P < 0.05). The phenomenon of arrest of G(1) phase was remarkable in Group A. The apoptotic rate of the cells of Group A was 47.21%, significantly higher than those of Groups B and C (14.18% and 3.36% respectively, both P < 0.05). An increased number of cells with chromosome condensation and fragmentation was found in Group A as compared with Groups B and C. CONCLUSION: The recombinant pEGFP-HPV18E6E7as can effectively inhibit the growth and proliferation of human cervical carcinoma HeLa cells, and further induce the cell apoptosis. The antisense RNA technology is available and may provide a new way to gene therapy of the cervical carcinoma.