Resistance to the Antitumor Agent Gallium Nitrate in Human Leukemic Cells Is Associated with Decreased Gallium/Iron Uptake, Increased Activity of Iron Regulatory Protein-1, and Decreased Ferritin Production

Janine P Wereley,Christopher R Chitambar

doi:10.1074/jbc.272.18.12151

Janine P Wereley, Christopher R Chitambar

Open Access

https://doi.org/10.1074/jbc.272.18.12151

Copy DOI

Abstract

The mechanism of drug resistance to gallium nitrate is not known. Since gallium can be incorporated into ferritin, an iron storage protein that protects cells from iron toxicity, we investigated whether ferritin expression was altered in gallium-resistant (R) CCRF-CEM cells. We found that the ferritin content of R cells was decreased, while heavy chain ferritin mRNA levels and iron regulatory protein-1 (IRP-1) RNA binding activity were increased. IRP-1 protein levels were similar in gallium-sensitive (S) and R cells, indicating that R cells contain a greater proportion of IRP-1 in a high affinity mRNA binding state. 59Fe uptake and transferrin receptor expression were decreased in R cells. In both S and R cells, gallium inhibited cellular 59Fe uptake, increased ferritin mRNA and protein, and decreased IRP-1 binding activity. Gallium uptake by R cells was markedly diminished; however, the sensitivity of R cells to gallium could be restored by increasing their uptake of gallium with excess transferrin. Our results suggest that R cells have developed resistance to gallium by down-regulating their uptake of gallium. In parallel, iron uptake by R cells is also decreased, leading to changes in iron homeostasis. Furthermore, since gallium has divergent effects on iron uptake and ferritin synthesis, its action may also include a direct effect on ferritin mRNA induction and IRP-1 activity.

Highlights

Gallium nitrate, a group IIIa metal salt with antineoplastic activity [1], is currently undergoing evaluation as a chemotherapeutic agent
Based on the concentration of gallium required to inhibit cell growth by 50%, R cells were ϳ8-fold more resistant to growth inhibition by gallium nitrate than S cells (Fig. 1)
Our studies revealed that whereas ferritin mRNA levels were increased in R cells, ferritin protein content was markedly diminished

Summary

The abbreviations used are

Transferrin; H, heavy chain; L, light chain; IRP, iron regulatory protein; IRE, iron-responsive element; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; S cells, gallium-sensitive cells; R cells, gallium-resistant cells. Under conditions of intracellular iron excess, IRPs are converted to low affinity forms that no longer bind IREs. As a result, ferritin synthesis proceeds unhindered in iron-repleted cells, and Tf receptor synthesis is down-regulated. Our studies show that the development of gallium resistance is due to a down-regulation in the transport of gallium into cells and that iron transport is affected in parallel, resulting in changes in the regulation of ferritin gene expression.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION