RESUMO
As a validated therapeutic target in several human cancers, the EGF receptor (EGFR) provides a focus to gain deeper insights into cancer pathophysiology. In this study, we report the identification of a naturally occurring and widely expressed EGFR isoform termed EGFRvA, which substitutes a Ser/Thr-rich peptide for part of the carboxyl-terminal regulatory domain of the receptor. Intriguingly, EGFRvA expression relates more closely to histopathologic grade and poor prognosis in patients with glioma. Ectopic expression of EGFRvA in cancer cells conferred a higher invasive capacity than EGFR in vitro and in vivo. Mechanistically, EGFRvA stimulated expression of STAT3, which upregulated heparin-binding EGF (HB-EGF). Reciprocally, HB-EGF stimulated phosphorylation of EGFRvA at Y845 along with STAT3, generating a positive feedback loop that may reinforce invasive function. The significance of EGFRvA expression was reinforced by findings that it is attenuated by miR-542-5p, a microRNA that is a known tumor suppressor. Taken together, our findings define this newfound EGFR isoform as a key theranostic molecule.
Assuntos
Receptores ErbB/fisiologia , Invasividade Neoplásica/genética , Neoplasias/patologia , Animais , Movimento Celular/genética , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Neoplasias/genética , Prognóstico , Isoformas de Proteínas/fisiologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Análise de Sobrevida , Células Tumorais CultivadasRESUMO
AIM: To express capsid tail protein P11 of T7 bacteriophage and produce mouse monoclonal antibody(mAb) against the protein. METHODS: P11 protein was cloned and recombinant P11 protein was expressed as a fusion protein with an N-terminal 6-His tag. The purified protein was used to immunize BALB/c mouse. The specificity of mAb was analyzed by ELISA and Western blot. RESULTS: P11 protein was successfully expressed and purified. SDS-PAGE analysis showed that the molecular weight of the expressed protein was approximately 27 kd. One hybridoma cell (2G11) secreting mAb against P11 was developed. The isotype of the mAb was IgG(2b). ELISA detection showed that titers of mAb was 1:8. 1 x 10(5) in ascites.Western blot analysis proved mAb obtained could react specifically to the recombinant p11 protein. CONCLUSION: Recombinant P11 protein and mAbs were successfully prepared.
Assuntos
Anticorpos Monoclonais , Bacteriófago T7/química , Proteínas do Capsídeo/imunologia , Imunoglobulina G/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Bacteriófago T7/genética , Fusão Celular , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Vetores Genéticos , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Virais/isolamento & purificaçãoRESUMO
AIM: To establish a cell line stably expressing EGFRvIIIex (epidermal growth factor receptor variant III extracellular domain) and evaluate its immunogenicity. METHODS: NIH3T3 cells stably expressing EGFRvIIIex was obtained by screening NIH3T3 cells transfected with pLNCX2-EGFRvIIIex, a plasmid encoding EGFRvIIIex. The expression level of EGFRvIIIex was examined by immunohistochemical staining and Western blot. The cell clone with the highest expression of EGFRvIIIex named as 3T3-vIIIex was used to immunize BALB/c mice. The titer and specificity of murine antiserum was evaluated by ELISA, Western blot and immunofluorescent staining. RESULTS: A NIN3T3 stable cell line with high EGFRvIIIex expression was obtained. The titer of the antiserum against human EGFRvIIIex was about 10(-5). Western blot and immunofluorescent staining analysis revealed the high specificity of the antiserum. CONCLUSION: High titer of antiserum against EGFRvIIIex can be obtained by NIH3T3 cell lines stably expressing EGFRvIIIex.