Mechanisms of neuroendocrine differentiation of lung carcinoma cells and their cell biological siginifcances : trials from the points of proteomics analyses

本研究课题的主要研究内容包括：1.分析用肺神经内分泌主基因hASH 1，2转染的人肺癌细胞系中表达的mRNA和蛋白质的改变。分析了神经内分泌基因的获得或缺失对细胞的生物学效应，包括细胞分化、增殖和形态发生的影响。本研究通过DNA微阵列和蛋白质组学分析发现的分子中的一些关键分子，并对这些分子的功能进行了研究，建立了转染hASH 1的人肺腺癌细胞系和转染Notch 1的人小细胞癌细胞系。HASH 1转染诱导腺癌细胞中的神经内分泌表型和神经内分泌标志物如嗜铬粒蛋白A的上调，但是，在裸鼠皮下组织中生长的肿瘤显示通常的腺癌形态，即使它们具有神经内分泌分化。另一方面，Notch 1转染的人小细胞癌细胞系 ...更多信息 丧失神经内分泌表型，表现为HES 1核转位和核hASH 1缺失。Notch 1转染后，培养细胞在转染前漂浮于培养液中，可粘附于培养皿上，并上调E-cadherin、integrin和CD 44的表达。此外，我们研究了Notch 1-Hes 1-hASH 1系统与其他细胞信号系统之间的串扰。细胞增殖信号通过MAP激酶和ATT/PI 3激酶下调hASH 1并改变细胞向非神经内分泌分化。我们发现小细胞癌细胞中存在大量的STAT 3，而非小细胞癌细胞中存在磷酸化的STAT 3，但STAT 3磷酸化不足。因此，neuroednodrine分化受到各种信号系统的影响。我们最近建立了Flag-、HA-、hASH 1-转染的人腺癌细胞系，以研究与hASH 1及其DNA结合位点相关的蛋白质。在不久的将来，我们将阐明参与神经内分泌分化的重要分子，以及更精确的肺上皮细胞和肺癌细胞分化的分子机制。少

Aims of this research projects include following issues : 1. analyses alterations of mRNAs and proteins expressed in human lung cancer cell lines transfected with a lung neuroendocrine master gene, hASH1, 2. analyses cell biological effects including cell differentiation, proliferation and morphogenesis by gain or loss of the neuroendocrine genes, 3. focuses some key molecules among the molecules found by DNA miroarray and proteomics analyses ad study functions of these molecules.We have established hASH1-transfected human lung adenocarcinoma cell lines and Notch1-transfected human small cell carcinoma cell lines. HASH1 transfection induced neuroendocrine phenotypes in adenocarcinoma cells and upregulation of neuroendocrine markers such as chromogranin A, but, the tumors grown in the subcutaneous tissue of nude mice showed usual adenocarcinoma morphology even though they have neuroendocrine differentiation. On the other hand, the Notch1-transfected human small cell carcinoma cell lines … More lose neuroendocrine phenotypes, showing nuclear translocation of HES1 and loss of nuclear hASH1. Moreover, Notch1 transfection made the cuture cells, which were floating in the medium before transfection, adhere to the plastic dishes, and up-regulated expression of E-cadherin, integrins, and CD44. Moreover, we studied cross-talks between the Notch1-Hes1-hASH1 system and other cell signalling systems. Cell proliferation signals via MAP kinase and ATT/PI3kinase downregulated hASH1 and alter cell differentiation toward non-neuroendocrine. We found that small cell carcinoma cells posesse large amount of STAT3, but STAT3 was underphosphorylated though non-small cell carcinoma cells have phosphorylated STAT3. Thus, neuroednodrine differentiation is affected by various signalling systems. We recently established Flag-, HA-, hASH1-transfected human adenocarcinoma cell lines to study proteins associated with hASH1 and its DNA binding sites. In the near future, we will clarify important molecules involved in neuroendocrine differentiation, and more precise molecular mechanisms of lung epithelial cell and lung carcinoma cell differentiation. Less