Nasopharyngeal carcinoma (NPC) poses serious health problems in Southern China and yet the molecular mechanism of the carcinogenesis remains unclear. We used modern proteomic technologies to compare the protein expression profiles between the NPC cell lines (HNE1 and CNE1 ) and an immortalized nasopharyngeal epithelial cell line NP69 to identify cancer related proteins. Cell lysates were separated by two-dimensional gel electrophoresis (2 DE ) and analyzed by PDQuest software. The differentially expressed proteins were identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). We discovered 15 up-regulated proteins and 18 down-regulated proteins in both HNE1 and CNE1 cell lines compared with NP69. These proteins are correlative with various functions, such as cell proliferation, apoptosis, cancer metastasis, metabolism, cytoskeleton and signal transduction. Western blotting analyses were further carried out to verify the differential expression of individual proteins. Several identified proteins in our research might be used as potential molecular markers to understand the molecular mechanism of NPC development and metastasis, and might be used as candidate targets for NPC treatments.

A subcellular proteomic method was applied to investigate the protein expression profiles of nasopharyngeal carcinoma (NPC) cell lines,CNE1 and CNE2,at various differentiation levels.Plasma membrane (PM) proteins were obtained by Percoll density grade centrifugation and subjected to twodimensional electrophoresis (2DE) followed by PDQuest software analysis.Nine proteins expressed with more than two folds difference were identified by MALDI-TOF-TOF,of which functions involved in cell differentiation,signal transduction,and metabolism.Half of these proteins,such as galectin-1 and annexin Ⅱ,were analyzed with real-time quantitative PCR or Westem blotting.We have tested a proteomic method to study differentiated NPCs at different levels and found several proteins that might be related to their biological characteristics.

To generate a mWAP-hLF hybrid locus that the transcription of human lactoferrin (hLF) genomic sequence is directed by the up & down stream regulatory sequence of murine whey acidic protein (mWAP) gene locus, we describe here a successive three-step 'Gap-repair' method. First, a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms was constructed. Then using 'Gap-repair 'method mediated by Red recombination system of lambda-prophage in Escherichia coli, in the first step, the 8 kb 3' flanking region of the mWAP gene was subcloned from the Bacterial artificial chromosome which harbors the mWAP gene locus(mWAP BAC) into the gap-repair vector; in the second step, the 29 kb hLF genomic sequence from the ATG code to the TAA code was subcloned from the hLF BAC; in the third step, the 12 kb 5' flanking region of the mWAP gene was subcloned from the mWAP BAC. Finally, all these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a 49 kb mWAP-hLF hybrid locus that the hLF genomic sequence was flanked by the 5' & 3' flanking region of mWAP gene locus was constructed. The result was confirmed by PCR, restriction enzyme digestion and sequencing. Our method provide a new way for the construction of large mammary-gland expression vector.
Animals ; Bioreactors ; DNA Repair ; genetics ; Genetic Engineering ; methods ; Humans ; Hybridization, Genetic ; Lactoferrin ; genetics ; Mammary Glands, Animal ; metabolism ; Mice ; Mice, Transgenic ; Milk Proteins ; genetics