RESUMO
Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Formaldeído/química , Neoplasias Pulmonares/metabolismo , Inclusão em Parafina , Análise Serial de Proteínas/métodos , Fixação de Tecidos , Western Blotting , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/patologia , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Coloração e RotulagemRESUMO
Mutations in the gene encoding dysferlin (DYSF) cause the allelic autosomal recessive disorders limb girdle muscular dystrophy 2B and Miyoshi myopathy. It encompasses 55 exons spanning 150 kb of genomic DNA. Dysferlin is involved in membrane repair in skeletal muscle. We identified three families with novel sequence variants in DYSF. All affected family members showed limb girdle weakness and had reduced or absent dysferlin protein on immunohistochemistry. All exons of DYSF were screened by genomic sequencing. Five novel variants in DYSF were found: two missense mutations (c.895G>A and c.4022T>C), one 5' donor splice-site variant (c.855+1delG), one nonsense mutation (c.1448C>A), and a variant in the 3'UTR of DYSF (c.*107T>A). All alterations were confirmed by restriction enzyme analysis and not found in 400 control alleles. Nonsense mediated RNA decay or changes in the three-dimensional protein structure resulting in intracellular dysferlin aggregates and finally the lack of dysferlin protein were identified as consequences of the novel DYSF variants.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas Musculares/química , Proteínas Musculares/genética , Distrofias Musculares/genética , Mutação , Adulto , Sequência de Aminoácidos , Análise Mutacional de DNA , Disferlina , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Proteínas de Membrana/deficiência , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/deficiência , Músculo Esquelético/patologia , Distrofias Musculares/diagnóstico , Linhagem , Dobramento de Proteína , Estrutura Terciária de Proteína , Sítios de Splice de RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
Extraocular muscles (EOM) are typically spared in Duchenne muscular dystrophy. We hypothesized that this might be due to different patterns of utrophin expression. The expression of utrophin was examined in EOM of normal cats using immunohistochemical methods and Western blot. For detecting acetylcholine receptors (AChR), we used alpha-bungarotoxin. Surprisingly, alpha-bungarotoxin failed to stain the AChR and no expression of utrophin could be detected at the neuromuscular junctions. Our study could indicate that the expression of utrophin is dependent on the structure of the AChR.