Cellular adaptation to down-regulated iron transport into lymphoid leukaemic cells: effects on the expression of the gene for ribonucleotide reductase

Abstract

Ribonucleotide reductase is an iron-containing enzyme that is essential for DNA synthesis. Whereas previous studies have used various iron chelators to examine the relationship between cellular iron metabolism and ribonucleotide reductase activity in cells, they have not elucidated the relationship between iron transport into cells and the expression of the gene for ribonucleotide reductase. To investigate this, we examined ribonucleotide reductase mRNA, protein and enzyme activity in a novel line of CCRF-CEM cells (DFe-T cells) that display an approx. 60% decrease in their uptake of iron compared with the parental wild-type cell line. We found that DFe-T cells displayed an approx. 40% decrease in ribonucleotide reductase specific enzyme activity relative to wild-type cells without a change in their proliferation. Kinetic analysis of CDP reductase activity revealed an approx. 60% decrease in V(max) in DFe-T cells without a change in K(m). Despite the decrease in enzyme activity, the mRNA and protein for the R1 and R2 subunits of ribonucleotide reductase in DFe-T cells were similar to those of wild-type cells. ESR spectroscopy studies revealed that DFe-T cells had a 22% decrease in the tyrosyl free radical of the R2 subunit, suggesting that a larger amount of R2 protein was present as functionally inactive apo-R2 in these cells. Our studies indicate that ribonucleotide reductase activity in CCRF-CEM cells can be down-regulated by more than 50% in response to down-regulated iron transport without an adverse effect on cell proliferation. Furthermore, our studies suggest a regulatory link between ribonucleotide reductase activity and iron transport into these cells.