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1.
Einstein (Säo Paulo) ; 20: eAO6450, 2022. tab, graf
Article in English | LILACS | ID: biblio-1364794

ABSTRACT

ABSTRACT Objective To understand the feasibility of FGFR3 tests in the Brazilian public health context, and to sample the mutational burden of this receptor in high-grade muscle invasive bladder cancer. Methods A total of 31 patients with high-grade muscle-invasive bladder cancer were included in the present study. Either transurethral resection of bladder tumor or radical cystectomy specimens were analyzed. Formalin-fixed paraffin-embedded tissue blocks were sectioned, hematoxylin and eosin stained, and histologic sections were reviewed. Total RNA was extracted using the RNeasy DSP formalin-fixed paraffin-embedded kit. Qualitative results were displayed in Rotor-Gene AssayManager software. Results Six patients were excluded. From the samples analyzed, four (16.7%) were considered inadequate and could not have their RNA extracted. Two patients presented FGFR3 mutations, accounting for 9.5% of material available for adequate analysis. The two mutations detected included a Y373C mutation in a male patient and a S249C mutation in a female patient. Conclusion FGFR3 mutations could be analyzed in 84% of our cohort and occurred in 9.5% of patients with high-grade muscle invasive bladder cancer in this Brazilian population. FGFR3 gene mutations are targets for therapeutic drugs in muscle-invasive bladder cancer. For this reason, know the frequency of these mutations can have a significant impact on public health policies and costs provisioning.


Subject(s)
Urinary Bladder Neoplasms/genetics , Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/metabolism , Carcinoma, Transitional Cell/pathology , Receptor, Fibroblast Growth Factor, Type 3/genetics , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Brazil , RNA , Prevalence , Eosine Yellowish-(YS) , Hematoxylin , Muscles/metabolism , Muscles/pathology , Mutation
2.
Yonsei Medical Journal ; : 831-839, 2016.
Article in English | WPRIM | ID: wpr-26893

ABSTRACT

PURPOSE: To examine the usefulness of various receptor tyrosine kinase expressions as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients. MATERIALS AND METHODS: We retrospectively analyzed the data of 98 patients with muscle invasive UC who underwent radical cystectomy between 2005 and 2010 in Yonsei Cancer Center. Using formalin fixed paraffin embedded tissues of primary tumors, immunohistochemical staining was done for human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 3 (FGFR3). RESULTS: There were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, significantly shorter median time to recurrence (TTR) (12.9 months vs. 49.0 months; p=0.008) and overall survival (OS) (22.3 months vs. 52.7 months; p=0.006) was found in patients with FGFR1 overexpression. By contrast, there was no difference in TTR or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% confidence interval: 1.27-3.90, p=0.006) in multivariate analysis. CONCLUSION: Our result showed that FGFR1 expression, but not FGFR3, is an adverse prognostic factor in muscle invasive UC patients after radical cystectomy. FGFR1 might be feasible for prognosis prediction and a potential therapeutic target after thorough validation in muscle invasive UC.


Subject(s)
Adult , Aged , Aged, 80 and over , Carcinoma/metabolism , Cystectomy , Female , Humans , Male , Middle Aged , Multivariate Analysis , Muscles/pathology , Neoplasm Invasiveness , Prognosis , Proportional Hazards Models , Receptor, ErbB-2/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Retrospective Studies , Survival Rate , Urinary Bladder Neoplasms/metabolism , Urothelium/pathology
3.
Electron. j. biotechnol ; 18(4): 302-306, July 2015. ilus
Article in English | LILACS | ID: lil-757868

ABSTRACT

Background Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies focus on the potential usage of therapeutic antibodies against FGFR3. Results In this study, a novel single-chain Fv (ScFv) against FGFR3 was prepared and characterized. To achieve the soluble expression, ScFv was fused with Sumo (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and cloned into pET-20b. The recombinant bacteria were induced by 0.5 mM Isopropyl-ß-d-thiogalactopyranoside (IPTG) for 16 h at 20°C, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After being cleaved by the Sumo protease, the recombinant ScFv was released from the fusion protein, and further purified by Ni-NTA chromatography. The purity of ScFv was shown to be higher than 95% and their yield reached 4 mg per liter of bacterial culture. In vitro data showed that ScFv can significantly attenuate FGF9-induced phosphorylation of FGFR3. Conclusion We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli.


Subject(s)
Receptor, Fibroblast Growth Factor, Type 3/metabolism , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/metabolism , Solubility , Mass Spectrometry , Recombinant Proteins , Blotting, Western , Escherichia coli
4.
Article in English | WPRIM | ID: wpr-634896

ABSTRACT

Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.


Subject(s)
Base Sequence , Bone Marrow Cells/cytology , Cartilage/cytology , Cell Differentiation/genetics , Cells, Cultured , Cloning, Molecular , Genetic Vectors/genetics , Molecular Sequence Data , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , SOX9 Transcription Factor/biosynthesis , SOX9 Transcription Factor/genetics , Stem Cells/cytology , Stromal Cells/cytology , Transfection
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