A study of adhesion ability of H-ras active cells to Fibronectin

Ras protein regulates cell signaling, proliferation and differentiation. Its expression increases in embriogenesis, healing of wounds and mitosis.Fibronectin, an extracellular matrix glycoprotein with a high molecular weight, binds to the specific integrin molecules on the cell membrane and plays an important role in many cellular activities, such as cell growth, tissue formation and apoptosis.In this study, rat embrio fibroblast cells (5PR7) were used. Growth curve of them was investigated by using trypan blue dying method. The adhesion capacity of 5RP7 to fibronectin was determined by adhesion assay (MTT). DNA fragmentation was used to determine the role of caspase enzymes in apoptosis in different concentrations of fibronectin.The growth rate of 5RP7 cells was found time concentration-dependent and the optimum number of cells for further studies is determined as 12.5x104. The cells adhered to 30 ?g/ml fibronectin maximally. They did not undergo apoptosis. The 5RP7 cells which adhered to 10 ?g/ml fibronectin underwent apoptosis and their appearance was similar to that of apoptotic DNA markers.Effects of caspase-3 and caspase-9 on apoptosis of 5RP7 cells that adhered to fibronectin were studied by addition of specific inhibitors of these enzymes. It is found that inhibition of these enzymes reduces the rate of DNA fragmentation in these cells. It is found that their apoptosis mechanism depends on the increase in the caspase-12 and then caspase-9 activities triggered by mitochondrial pathway or increase in [Ca]+2 that adhered to endoplasmic reticulum.