Investigation of MMP-2 and -9 in a Highly Invasive A431 Tumor Cell Sub-line Selected from a Boyden Chamber Assay

In this study, highly invasive tumor cell lines (designated A431-I, -II and -III) derived from parental A431 tumor cells (A431-P) w ere isolated by three successive passages through a Boyden chamber with matrigel-coated membrane support. The invasive potential and the activity of secreted MMP-9 of each sub-line increased significantly compared to the A431-P (A431-III > A431-II > A431-I) as evidenced by the in vitro invasion assay, gelatin zymography and immunoblotting analyses. RT-PCR results also revealed the elevated expression of MMP-9 in A431-III. We further characterized the A431-III sub-line and found these cells exhibited a greater potential for attachment and spreading on fibronectin-coated substratum and for migration. The A431-III cells displayed multiple cytoplasmic extensions with focal contacts (vinculin-positive staining) during cell spreading within 30 min. We also noticed an increase in FAK phosphorylation, but no significant change in FAK protein level in the A431- III sub-line compared to those of A431-P cells. Together, these results demonstrate that the greater invasion potential exhibited by the highly invasive A431-III subline is likely attributed to an increased ability for attachment, spreading and migration, as well as increased MMP activity. Thus, A431-P and the highly invasive A431-III sub-line could be an excellent model for studying the mechanism of cancer metastasis. Migration of tumor cells from the primary neoplastic clone into surrounding normal tissues is a process generally called invasion. Remodeling of the extracellular