RESUMO
Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is found predominantly on endothelial cells. The purpose of this study was to determine the role of VEGFR2 expression in NSCLC cells. NSCLC cells and tissue sections were stained for VEGFR2 expression by immunohistochemistry (IHC). Immunoblotting and ELISA were used to determine the activation and inhibition of VEGFR2 and its downstream signalling pathways. Five-day proliferation assays were carried out in the presence or absence of VEGF. IHC analysis of NSCLC demonstrated tumour cell VEGFR2 expression in 20% of samples. Immunoblot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation was apparent in NSCLC cells, and was associated with increased tumor cell proliferation. Cediranib treatment or siRNA against VEGFR2 inhibited VEGF-dependent increases in cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine. Our data suggest that a subset of NSCLC tumour cells express functional VEGFR2 which can act to promote VEGF-dependent tumour cell growth. In this tumour subset, therapies targeting VEGFR2 signalling, such as cediranib, have the potential to inhibit both tumour cell proliferation and angiogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Benzimidazóis , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologiaRESUMO
An increased incidence of psychiatric and structural brain abnormalities in individuals with Klinefelter syndrome (KS, 47 XXY) could be due to the presence of extra copies of X-Y homologous genes that escape X inactivation. Of particular interest are the two brain-expressed genes Protocadherin11XY (PCDH11XY) and the Synaptobrevin-like gene (SYBL1) which have been duplicated from the X chromosome to the Y chromosome to give X-Y homologous gene pairs that are specific to modern humans. We examined the DNA of KS individuals reported recently by DeLisi et al. 2005 and determined the parental origin of the X alleles, the degree of skewed X inactivation and investigated the CpG island methylation status of PCDH11XY and SYBL1 by bisulphite sequencing and quantification of methylated HpaII sites. We used a novel method for quantification of unmethylated CpGs with the restriction enzyme McrBC which cuts methylated but not unmethylated CpGs. The results showed that KS individuals have two methylated and one unmethylated SYBL1 allele whereas PCDH11XY is unmethylated and escapes X inactivation on the extra X chromosome. Overexpression of PCDH11XY in KS is probable and variable escape from inactivation of this Homo sapiens-specific gene could account for some abnormalities in KS. The origin of the parental alleles or their preferential X inactivation was not associated with psychotic symptoms.
Assuntos
Caderinas/genética , Metilação de DNA , Síndrome de Klinefelter/genética , Proteínas R-SNARE/genética , Alelos , Ilhas de CpG/genética , DNA/genética , DNA/metabolismo , Saúde da Família , Feminino , Genótipo , Humanos , Cariotipagem , Masculino , Protocaderinas , Inativação do Cromossomo XRESUMO
OBJECTIVES: The genetic basis of schizophrenia is obscure. In an XX male patient with schizophrenia we previously showed that one X;Y translocation breakpoint was in pseudoautosomal region 1 (PAR1) with the effect that the proximal segment of PAR1 from the PAR1 boundary to acetylserotonin N-methyl transferase (ASMT) distally was triplicated in this patient. This study determined whether dosage imbalances of X-Y homologous regions in general are associated with schizophrenia. METHODS: A multiplex semi-quantitative polymerase chain reaction assay was developed to quantify MIC2 gene as a representative of PAR1 and compare it with the SYBL1 gene which maps in pseudoautosomal region 2 (PAR2) and protocadherin XY (PCDHXY), located at Xq21.3. Each of these three loci was co-amplified with the autosomal gene MSX2 using Cy5-labelled primers and the products separated by electrophoresis in polyacrylamide gels. Results were expressed as ratios of peak area of the target gene to MSX2 which served as an internal dosage control. RESULTS: Using genomes with sex chromosome aneuploidies, the method was found sensitive enough to detect a two-fold difference in gene copy number. We confirmed the MIC2 triplication in the XX male patient but found no significant difference in gene dosage of MIC2, PCDHXY and SYBL1 in a panel of 17 patients with schizophrenia compared to controls. CONCLUSIONS: No evidence was obtained for gene dosage imbalances in MIC2, PCDHXY and SYBL1 in patients with schizophrenia.
