Selection of variant hepatoma cells in liver-specific growth media: regulation at the mRNA level

Abstract

Abstract Three liver-specific growth media, respectively free of arginine (Arg -), tyrosine (Tyr -) and glucose (G -), have been used to characterize cells of the rat H4IIEC3, human HepG2 and mouse BW hepatoma lines. Cells of clone FaO, a derivative of line H4IIEC3, freely grew in Tyr - and G - media, and gave rise to stable variants in Arg - conditions. Cells of line HepG2 and clone BWTG3, a derivative of line BW, degenerated in all three media. Arg and tyr variants were however derived from HepG2 cells; their genesis appeared to be pathway specific, illustrating the complexity of the regulatory loops that are implicated in the control of the differentiated state. No variant was ever obtained with BWTG3 cells, demonstrating the stability of their deficiency in the post-natal hepatic functions that are involved in Arg -, Tyr - and G - selections. Variant clones of HepG2 and FaO cells that have been isolated in Arg - medium were characterized in details for liver-specific urea-cycle enzyme activities and mRNA. These variants were shown to be controlled at the mRNA level, most likely at transcription. Isolation of stable FaO and HepG2 variant clones as well as the converse demonstration of the stable deficiency of BWTG3 cells in postnatal hepatic functions were aimed at expression cloning. Our results are thus discussed in terms of transfection with full-length cDNA expression libraries and cloning of regulatory genes that could activate or extinguish liver specific genes.