Ras-transfection up-regulated HaCaT cell migration: Inhibition by Marimastat

Cell migration is an essential process in physiological and pathological co nditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present st udy, the migration of two HaCaT-ras clones (metastatic or not), was compare d with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV c ollagen was similar for primary cultured keratinocytes and HaCaT, whereas i t was markedly higher for the HaCaT-ras clones. High motility of ras-transf ected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different ad hesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonst rating the important role of MMPs in the migration process. Under our exper imental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 acti vity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and i ts secretion was markedly higher for HaCaT-ras clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP- 2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with H aCaT. These results suggest that Ha-ras oncogene could be a stimulating fac tor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines.