In the test of SSH, a pair of high and low-metastatic monoclonal cell strains termed PLA801 D and PLA801 C both derived from the same parental lung giant cell carcinoma cell line PLA801 were used as model cells. The highly metastatic cell subpopulation, PLA801D acted as tester in SSH and the poorly metastatic cell subpopulation PLA801C was driver. Microarray was used to screen library generated by SSH efficiently. Some clones with high expressing level in the results of microarray had been confirmed to have high expressing by RT-PCR and Northern blot. 79 ESTs were found to be expressed two times or more in D than in C. Further bioinformatics analysis led to the discovery of one novel metastasis associated genes, mag-1 highlyidentical with BC006236 (the ID number of GenBank) .Sense and anti-sense orientation of mag-1 gene were inserted into the multiply cloning sites of pcDNA3. Sense construct was transfected into the poorly metastatic strain PLA-801C by the way of lipofection., and anti-sense was sent into highly metastasis strain PLA-801D respectively. The resistant clones, with sense mag-1, construct was termed as c1, with anti-sense mag-1 as d1, were selected by G418. In both MTT growth and colony formation assays, significant growth promotion was observed, a feature common to cells over-expressing the mag-1 gene. Consistently growth inhibition was also seen on d1. Cell cycle was analyzed with a flow cytometer, the result told us that the proliferation index (PI) of c1 was higher than mock control. In the experiment of invasion assay by method of Trans-well, we found that mag-1 could impact the invasion ability of PLA-801C/D cell remarkably without changing the cells’ character of migration. Cells with sense constructs became more easily to adhere to extracellular matrix (ECM); while cells with anti-sense construct the ability of ECM adhesion was partly inhibited. The expression of related tumor marker such as p53, MMP-2, CD44s and PCNA in transfected cells were also examined by using RT-PCR and Western Blot analysis. In cell c1 the proteins quantity of CD44s, MMP-2 and mRNA level of PCNA were elevated; CD44s was down regulated in d1. The free intracellular [Ca2+] of all transfected cells were examined by confocal microscope under the laser of 464nm. In this experiment, free [Ca2+] of cytoplasm was found to be decreased remarkably in cell d1. In summary, mag-1 gene had impacts on the metastasis character of human lung-giant-cell carcinoma cell line PLA-801(C/D) in several steps including proliferation, adhesion, invasion etc. mag-1 gene also had the ability of regulating the expression of MMP-2, PCNA, CD44 and p53 genes. All these suggested that mag-1 imposes on tumor metastasis probably by the mechanism of interacting with MMP-2 and/or CD44s.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]