Inductive effect of copper deficiency on the reversion of dedifferentiated rat hepatoma cells and on gene amplification

Abstract

Cells of a dedifferentiated rat hepatoma clone were submitted in vitro to copper deficiency. This treatment caused inhibition of cell growth. In addition, in treated cultures, the frequency of differentiated revertants selected in glucose-free medium was drastically increased when compared with the spontaneous frequency. The maximum effect was observed when cell proliferation spontaneously resumed after 20 days of copper deficiency. Furthermore, a copper depletion/replenishment protocol applied before the selection of revertants reduced the period of time of copper deficiency that was necessary to provoke the reversion process. It has been previously demonstrated that cell growth arrest and reinitiation may induce gene amplification events. Amplification of the dihydrofolate reductase gene as an indicator of such events was tested during the copper deficiency treatment. The frequency of cells resistant to increasing methotrexate concentrations due to gene amplification was enhanced by the treatment, just as was the frequency of differentiated revertants. These results suggest that in rat hepatoma cells the phenotypic transition to the stable differentiated state involves gene amplification and/or genome rearrangement.