Background: Large cell neuroendocrine carcinoma (LCNEC) shows poor prognosis due to aggressive growth and resistance to chemotherapy/radiotherapy. Few cell lines of LCNEC are available for the screening of chemotherapeutic agents and investigation of the biological characteristics. In order to increase the knowledge on LCNEC, we established and analyzed a new cell line TMU-1 from the primary LCNEC of the lung. Material and Methods: TMU-1 was established by the method previously described. Briefly, excised tumor tissue was minced and incubated in the medium without serum at first. After selection, only tumor cells were incubated in the medium containing 10% FCS. DNA was extracted and investigated by Agilent human aCGH microarray. Genomic data was analyzed by CGH analytic software to detect either amplified or deleted sites of chromosomes. For proteome analysis, harvested cells were processed and separated into cytoplasm fraction and membrane fraction at first. Then proteins were extracted from each fraction, alkylated and digested by trypsin. Resulted peptide mixtures were subjected to the LTQ-Orbitrap high resolution mass spectrometry following a micro-flow liquid chromatography separation (\#956;LC-MS/MS) interfaced with a nano-electrospray ionization source. Identification of peptides/proteins was carried out using the MASCOT software for database search. Results: TMU-1 shows biphasic growth pattern, sticky type and floating type in the media having endocrine granules in the cytoplasm. Neuroendocrine markers, NCAM-1, synaptophysin and chromogranin A were immunehistochemically detected. There were 10 amplified sites on the whole genome. Especially, first, ninth and thirteenth were highly amplified. The amplified site of chromosome 1 contains MYCL1. While, 25 sites were deleted on the whole genome localized to some chromosomes. The deleted lesion of eleventh chromosome encodes P53AIP1 related to apoptosis. On the other hand, proteomic analysis identified about 900 proteins as a whole. 772 proteins were identified in sticky TMU-1 and 738 proteins were identified in floating TMU-1. Among them, only 168 proteins (92 in membrane, 76 in cytoplasm) were specific for sticky TMU-1 and 138 proteins (79 in membrane, 59 in cytoplasm) were specific for floating TMU-1. Interestingly, NCAM-1 was always expressed except in the membrane of floating type TMU-1. We could found only nine proteins with genomic amplification. Conclusions: We established a new cell line of LCNEC, TMU-1 which has two morphological types and different patterns of expression of proteins according to each shape. We also found huge discrepancy between genomic amplification and proteomic expression in TMU-1. We are further investigating TMU-1 by using the techniques of glyco-proteomics and small RNA to explain the discrepancy and to identify specific therapeutic targets to improve the clinical outcome.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5264.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO