Profiling analysis of differential gene expression between hematogenous and peritoneal metastatic sublines of human pancreatic cancer using a DNA chip.

We established the novel sublines HPC-1H5, HPC-3H4, HPC-4H4, and Panc-1H5, which have a high potential of liver metastasis, and HPC-1P5a, HPC-3P4a, HPC-4P4a, and Panc-1P5a, which have a high potential of peritoneal dissemination, derived from low metastatic HPC-1, HPC-3, HPC-4, and Panc-1cell lines, respectively. To clarify the molecular mechanisms of cancer metastasis and of the different levels of gene expression in a variety of metastatic potentials in pancreatic cancer, we performed a broad analysis of differential gene expression analysis between parental cell lines and metastatic sublines. In comparison with the parental cell lines, 65 and 36 genes were overexpressed and underexpressed in highly liver-metastatic sublines. On the other hand, 43 and 45 genes were overexpressed and underexpressed in highly peritoneal-metastatic sublines. uPAR and Serin protease were overexpressed, and E2A and IGF1R were underexpressed in both metastatic sublines. Hierarchical clustering analysis revealed 22 genes classifying liver, peritoneal metastatic sublines and low-metastatic parental cell lines. These genes might be targeted genes separating those two major metastatic forms after surgery. A greater number of cell line samples and more genes will have to be utilized in future studies in order to understand the involvement of genes in cancer metastasis more thoroughly. However, these results will help to clarify the molecular mechanisms of pancreatic cancer metastasis.

Metastatic-associated biological properties and differential gene expression profiles in established highly liver and peritoneal metastatic cell lines of human pancreatic cancer.

To elucidate metastasis mechanisms, we established a Panc-1H5 subline with a highly liver metastatic cell line and a Panc-1P4a with a highly peritoneal metastatic cell line, which were sequentially selected from the parental pancreatic cancer cell line Panc-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis with a cDNA macroarray. The tumorigenicity, motile activity, adhesive activity and cytokine production of metastatic sublines were higher than those of parental Panc-1 cells. Particularly, in Panc-1H5 cells, adhesive activity to the extracellular matrix and angiogenetic factors increased, whereas in Panc-1P4a cells, motile activity was extremely enhanced compared with Panc-1 cells. Histopathological findings for the three cell lines were the same. In cDNA macroarray analysis of Panc-1H5 cells, 11 genes were up-regulated and 20 genes were down-regulated compared with parental Panc-1 cells. In Panc-1P4a cells, 7 genes were up-regulated and 13 genes were down-regulated compared with parental Panc-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver and peritoneal metastasis and these results provide new insight into the study of human pancreatic cancer metastasis.

Analysis of nuclear localization signals using a green fluorescent protein-fusion protein library.

We describe here an efficient method for identifying intracellular localization signals in proteins with stereospecific intracellular localizations in culture cells. The method involves rapid fluorescence screening of cells transfected with a cDNA library in which cDNAs are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP). We analyzed nuclear localization and nuclear localization signals (NLSs) in a model application of this method. As a result, we identified classical NLSs in 75% of nuclear localized proteins. We identified some novel NLS candidates among the classical NLS-negative sequences whose nuclear localization was also identified in another cell line and with other molecular tag sequences. This method will be useful for identifying intracellular localization signals and for more detailed analysis of intracellular architecture.