Influence of a feeder layer in cell culture: modulation of the transcription factors Sp1 and Sp3 by irradiated 3T3 cells.

Abstract

Abstract: : Purpose:Primary cultured cells are used in both clinical and basic research. Human keratinocytes that are primarily used for basic research are often cultivated on plastic or on extracellular matrix–coated culture plates. However, when used either for transplantation purposes or clinical research, these cells are often co–cultured with lethally irradiated 3T3 cells as a feeder layer, as is the case for primary cultured human corneal epithelial cells (CHCEs). It is now recognized that the proliferative state of any given cell culture is determined by the level to which specific nuclear transcription factors are expressed in such cells, which, in turn, determine the expression of genes whose products are required in particularly important processes like cell migration and adhesion. Variations in the level of expression of the positive transcription factors Sp1 and Sp3 were recently reported to occur in corneal epithelial cells isolated from different human donors. The aim of the present study was to demonstrate the influence irradiated 3T3 cells might have on the expression and DNA binding affinity of Sp1 and Sp3 in primary cultured cells. Methods:As human skin keratinocytes from different histological origins can be obtained easily from many different donors, we selected these cells in order to examine how irradiated 3T3 cells can alter the expression and DNA binding of Sp1/Sp3. Both adult (Ad) and newborn (Nb) human skin keratinocytes were cultured with or without lethally irradiated 3T3 cells as a feeder layer. Crude nuclear extracts were prepared from both types of cells at each passage. The DNA binding ability of Sp1/Sp3 was monitored by electrophoretic mobility shift assays (EMSAs) whereas their expression at the protein level was examined by Western blotting. Results:Lethally irradiated 3T3 cells induced an increase in the level of expression of both Sp1 and Sp3. Although the basal, unstimulated level of Sp1/Sp3 may vary dramatically in an age–dependent manner in cultured skin keratinocytes obtained from different donors, all cells responded positively to the presence of irradiated 3T3 cells. Conclusions:Lethally irradiated 3T3 cells used as a feeder layer modulate both the expression and DNA binding affinity of Sp1 and Sp3 in vitro. This 3T3–mediated increase in the expression of Sp1/Sp3 also help maintain both Ad and Nb keratinocytes in a highly proliferative state and also delays their progression toward terminal differentiation.

Keywords: signal transduction • transcription factors • transplantation