Gene expression profiling of endobronchial ultrasound (EBUS)-derived cytological fine needle aspirates from hilar and mediastinal lymph nodes in non-small cell lung cancer.

OBJECTIVE: Endobronchial ultrasound (EBUS) allows minimally invasive sampling of hilar and mediastinal lymph nodes and has an established role in non-small cell lung cancer (NSCLC) diagnosis and staging. Molecular biomarkers are being explored increasingly in lung cancer research. Gene expression profiling (GEP) is a microarray-based technology that comprehensively assesses genome-wide changes in gene expression that can provide tumour-specific molecular signatures with the potential to predict prognosis and treatment responsiveness. We assessed the feasibility of using EBUS-derived aspirates from benign and tumour-infiltrated lymph nodes for GEP. METHODS: RNA was extracted from EBUS-directed transbronchial fine needle aspiration samples in routine clinical practice. GEP was subsequently performed in six patients with NSCLC, three of whom had tumour-infiltrated nodes and three who had benign lymph nodes; the differences in gene expression were then compared. RESULTS: RNA was successfully extracted in 29 of 32 patients, 12 of whom were diagnosed with NSCLC. RNA yield (median, 12.1 µg) and RNA integrity (median, 6.3) were sufficient after amplification for GEP. Benign and malignant nodes in adenocarcinoma were discriminated by principal component analysis and hierarchical clustering with different expression patterns between malignant and benign nodes. CONCLUSION: We have demonstrated the feasibility of RNA extraction and GEP on EBUS-derived transbronchial fine needle aspirates from benign and tumour-infiltrated lymph nodes in patients with known NSCLC in routine clinical practice. Further studies on larger patient cohorts are required to identify expression profiles that robustly differentiate benign from malignant lymph nodes in NSCLC.

Uptake of endocytic markers by rice cells: variations related to the growth phase.

Endocytosis is now considered a basic cellular process common to plant cells. Although both non-specific and receptor-mediated endocytosis appear to take place in plant cells, the physiological role of the latter remains unclear. We have investigated the endocytic process in rice cell suspensions using two biotinylated proteins, peroxidase and bovine serum albumin (bHRP and bBSA), as markers. First, we show that markers are internalized by rice cells and appear in intracellular membranes. The uptake of the two markers is temperature dependent, saturable with time and markers dose and it is competed by free biotin. Thus, it shows the properties of a receptor-mediated process. We also show that uptake of markers is strongly influenced by growth phase as optimal uptake occurs during the lag phase, but the initiation of the exponential growth phase decreases uptake drastically. Arrest of the cell cycle by starvation of either a nutrient (phosphate) or a growth regulator (2,4-dichlorophenoxyacetic acid), both components of the culture medium, does not modify the rate of bBSA uptake. Subsequent readdition of these components results in growth recovery and a dramatic decrease in bBSA uptake. On the other hand, nocodazole treatment, a method to arrest the cell cycle by microtubule depolymerization, inhibited bBSA uptake. The possible causes for this arrest of endocytosis are discussed.

Characterization of Cop I coat proteins in plant cells.

Membrane traffic in eukaryotic cells is mediated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been unequivocally demonstrated, although coated vesicles (probably with a COP coat) can be seen by electron microscopy. At the gene level, plant cells seem to contain all the components necessary to form COP-coated vesicles. In this paper, we have used antibodies raised against mammalian COPI coat proteins to detect putative homologues in rice (Oryza sativa) cells. Using these antibodies, we have found that rice cells contain alpha-, beta-, beta'-, and gamma-COP, as well as ADP-ribosylation factor (ARF) 1 protein. In addition, we show that antibodies against mammalian beta'-COP can immunoprecipitate not only beta'-COP but also alpha-, beta-, and gamma-COP, suggesting that COPI components in rice cells exist as a complex (or coatomer) in the cytosol, as in mammalian cells. Finally, we show that COP binding to membranes is GTP-dependent, and that ARF1 also binds to membranes in a GTP-dependent manner.