Identification of metal ion binding sites based on amino acid sequences.

The identification of metal ion binding sites is important for protein function annotation and the design of new drug molecules. This study presents an effective method of analyzing and identifying the binding residues of metal ions based solely on sequence information. Ten metal ions were extracted from the BioLip database: Zn2+, Cu2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Na+, K+ and Co2+. The analysis showed that Zn2+, Cu2+, Fe2+, Fe3+, and Co2+ were sensitive to the conservation of amino acids at binding sites, and promising results can be achieved using the Position Weight Scoring Matrix algorithm, with an accuracy of over 79.9% and a Matthews correlation coefficient of over 0.6. The binding sites of other metals can also be accurately identified using the Support Vector Machine algorithm with multifeature parameters as input. In addition, we found that Ca2+ was insensitive to hydrophobicity and hydrophilicity information and Mn2+ was insensitive to polarization charge information. An online server was constructed based on the framework of the proposed method and is freely available at http://60.31.198.140:8081/metal/HomePage/HomePage.html.

Contributions of lung tissue extracts to invasion and migration of human hepatocellular carcinoma cells with various metastatic potentials.

PURPOSE: The MHCC97 cell line contains two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potentials, for which the pulmonary metastatic rate was 100% vs 40% between the two human hepatocellular carcinoma (HCC) clones. In an effort to elucidate the mechanism of organ-specific metastasis, we studied the effect of lung extracts from C57BL/6 mice on migration and invasion using the MHCC97 cell line. METHODS: Determination of migration and invasion induced by lung extracts to MHCC97 cell lines was examined by chemoinvasion assay. The organization of cytoskeleton was tested using filamentous actin (F-actin) polymerization assay and flow cytometry. The activity of matrix metalloproteinase (MMPs) was analyzed by zymogram. Fluorescence double staining was employed for MMPs and F-actin colocalization in MHCC97-H cells induced by lung extracts. RESULTS: The number of cells in response to extracts of lung, liver, kidney, and spleen in MHCC97-H cells was 64+/-10, 6+/-2, 22+/-4, and 3+/-1, respectively. Of the extracts, lung extracts showed significant differences to promote the migratory and invasive ability for MHCC97-H cells( p<0.001). The number of cells in response to lung extracts was threefold higher in MHCC97-H than that of cells in MHCC97-L ( p<0.001). Confocal laser scan microscope of MHCC97-H cells and MHCC97-L cells stimulated with lung extracts revealed pseudopodia formation around the cell front at the indicated time point. With the time increasing, the pseudopodia formation became increasingly more obvious and distinct. Compared with unstimulated cells, analysis of FACS showed a transient 1.9-fold and 1.7-fold increase in F-actin within 30 s in MHCC97-H cells and MHCC97-L cells, respectively. Confocal laser scan microscopy of MHCC97-H cells stimulated in suspension with lung extracts revealed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. After the cells were incubated with lung extracts, not only expressions of active and latent form of MMP-9 were upregulated, but that of latent form of MMP-2 was increased in the MHCC97-H cells and MHCC97-L cells. The levels of latent and active form of MMP-9(18.8+/-1.2, 100.1+/-1.1), and latent form of MMP-2(22.4+/-1.3) were much higher in MHCC97-H cells than those of MHCC97-L cells, which were 7.8+/-0.3, 40.8+/-2.2, and 8.2+/-0.4, respectively. MMP-9 was mainly localized perinuclear pool when the MHCC97-H cells were incubated with serum-free medium. After the cells were stimulated with lung extracts, MMP-9 were expressed and colocalized with F-actin at the front of extending pseudopodia. CONCLUSIONS: Our results indicate that the expression of matrix metalloproteinase and the pseudopodia formation in MHCC97-H cells may correlate to the metastatic potential; thus, the host environment may contribute to the preferential metastasis of HCC cells to the lung depending on the high level of MMP-9 and migratory ability.

[Establishment of human hepatocellular carcinoma cell line with spontaneous pulmonary metastasis through in vivo selection].

OBJECTIVE: To establish a hepatocellular carcinoma (HCC) cell line from lung metastatic lesions of human HCC in nude mice so as to provide suitable model for the study of lung-metastasis-related molecular mechanisms. METHODS: HCC clone cells MHCC97-H were inoculated subcutaneously into nude mice. The pulmonary metastatic lesions were harvested from the moribund animals and then re-implanted into the nude mice for the second round of selection. The same procedure was repeated twice. New cell line from the third round of lung metastasis was thus established and the following parameters were studied: morphology, in vitro growth curve, plate efficiency, migration, karyotype, flow cytometry, immunocytochemistry for AFP, albumin and cytokeratin 8 (CK8), flourescent PCR for HBV DNA, tumorigenicity and spontaneous metastasis via subcutaneous injection and orthotopic implantation of tumor tissue. RESULTS: The cell line established from nude mouse lung metastasis was designated as HCCLM3, consisting of polygonal epithelial cells with hypotriploid karyotype, its main range of chromosome being 55 - 58. The cell population doubling time was 34.9 hours, plate efficiency 32.4 +/- 3.2%, and cell migration rate 20 +/- 2 microm/h. The cells were positive for AFP, albumin, CK8, and P16 and negative for P53, nm23 and HBsAg. Fluorescent PCR showed HBV DNA integration into cellular genome. When 5 x 10(6) cells were injected subcutaneously into nude mice, the tumorigenicity was 100% with a latency period of 11 +/- 1 d. Five weeks after subcutaneous injection, the pulmonary metastatic rate was 100%, the median number of lung metastases being 121 per mouse. After orthotopic implantation of tumor tissue into nude mouse liver for 35 d, wide spread regional and distant metastases occurred, the metastatic rate was 100% for abdominal wall, 80% for organs in abdominal cavity, 100% for liver, 70% for diaphragm, and 100% for lung. The median number of lung metastatic lesions was 268 per mouse. CONCLUSION: A new HCC cell line has been established, which is characterized by high pulmonary metastasis via both subcutaneous and orthotopic inoculation. It provides a new model for the study of liver cancer metastasis.