Iron transport in a lymphoid cell line with the hemochromatosis C282Y mutation.

The gene for hemochromatosis (HFE) is expressed in a variety of cells, including those not thought to be affected by this disease. The impact of HFE on iron transport was examined in B-lymphoid cell lines developed from a patient with hemochromatosis with the HFE C282Y mutation (C282Y cells) and an individual with the wild-type HFE gene (WT cells). Whereas both cell lines expressed HFE protein, C282Y cells displayed less HFE protein at the cell surface. Transferrin receptor (TfR) number was 2- to 3-fold greater in WT cells than in C282Y cells, while TfR affinity for transferrin (Tf) was slightly lower in C282Y cells. TfR distribution between intracellular and cell-surface compartments was similar in both cell lines. Iron uptake per cell was greater in WT cells but was not increased proportional to TfR number. When considered relative to cell-surface TfR number, however, iron uptake and Tf internalization were actually greater in C282Y cells. Surprisingly, Tf-independent iron uptake was also significantly greater in C282Y cells than in WT cells. The ferritin content of C282Y cells was approximately 40% that of WT cells. Exposure of cells to pro-oxidant conditions in culture led to a greater inhibition of proliferation in C282Y cells than in WT cells. Our results indicate that in this B-lymphoid cell line, the HFE C282Y mutation affects both Tf-dependent and -independent iron uptake and enhances cell sensitivity to oxidative stress. The role of HFE in iron uptake by B cells may extend beyond its known interaction with the TfR.

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

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.

Increased sensitivity of hydroxyurea-resistant leukemic cells to gemcitabine.

Tumor cell resistance to certain chemotherapeutic agents may result in cross-resistance to related antineoplastic agents. To study cross-resistance among inhibitors of ribonucleotide reductase, we developed hydroxyurea-resistant (HU-R) CCRF-CEM cells. These cells were 6-fold more resistant to hydroxyurea than the parent hydroxyurea-sensitive (HU-S) cell line and displayed an increase in the mRNA and protein of the R2 subunit of ribonucleotide reductase. We examined whether HU-R cells were cross-resistant to gemcitabine, a drug that blocks cell proliferation by inhibiting ribonucleotide reductase and incorporating itself into DNA. Contrary to our expectation, HU-R cells had an increased sensitivity to gemcitabine. The IC50 of gemcitabine was 0.061 +/- 0.03 microM for HU-R cells versus 0.16 +/- 0.02 microM for HU-S cells (P = 0.005). The cellular uptake of [3H]gemcitabine and its incorporation into DNA were increased in HU-R cells. Over an 18-h incubation with radiolabeled gemcitabine (0.25 microM), gemcitabine uptake was 286 +/- 37.3 fmol/10(6) cells for HU-R cells and 128 +/- 8.8 fmol/10(6) cells for HU-S cells (P = 0.03). The incorporation of gemcitabine into DNA was 75 +/- 6.7 fmol/10(6) cells for HU-R cells versus 22 +/- 0.6 fmol/10(6) cells for HU-S cells (P < 0.02). Our studies suggest that the increased sensitivity of HU-R cells to gemcitabine results from increased drug uptake by these cells. This, in turn, favors the incorporation of gemcitabine into DNA, resulting in enhanced cytotoxicity. The increased sensitivity of malignant cells to gemcitabine after the development of hydroxyurea resistance may be relevant to the design of chemotherapeutic trials with these drugs.

Transferrin receptor-dependent and -independent iron transport in gallium-resistant human lymphoid leukemic cells.

Recent studies showed that gallium and iron uptake are decreased in gallium-resistant (R) CCRF-CEM cells; however, the mechanisms involved were not fully elucidated. In the present study, we compared the cellular uptake of 59Fe-transferrin (Tf) and 59Fe-pyridoxal isonicotinoyl hydrazone (PIH) to determine whether the decrease in iron uptake by R cells is caused by changes in Tf receptor (TfR)-dependent or TfR-independent iron uptake. We found that both 59Fe-Tf and 59Fe-PIH uptake were decreased in R cells. The uptake of 59Fe-Tf but not 59Fe-PIH could be blocked by an anti-TfR monoclonal antibody. After 59Fe-Tf uptake, R cells released greater amounts of 59Fe than gallium-sensitive (S) cells. However, after 59Fe-PIH uptake 59Fe release from S and R cells was similar. 125I-Tf exocytosis was greater in R cells. At confluency, S and R cells expressed equivalent amounts of TfR; however, at 24 and 48 hours in culture, TfR expression was lower in R cells. Our study suggests that the decrease in Tf-Fe uptake by R cells is caused by a combination of enhanced iron efflux from cells and decreased TfR-mediated iron transport into cells. Furthermore, because TfR-dependent and -independent iron uptake is decreased in R cells, both uptake systems may be controlled at some level by similar regulatory signal(s).

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.

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.

Evaluation of transferrin and gallium-pyridoxal isonicotinoyl hydrazone as potential therapeutic agents to overcome lymphoid leukemic cell resistance to gallium nitrate.

Gallium nitrate is active against lymphoma and bladder cancer; however, little is understood about tumor resistance to this drug. Transferrin, the iron transport protein, increases gallium uptake by cells, whereas pyridoxal isonicotinoyl hydrazone (PIH), an iron chelator, transports iron into cells. Therefore, we examined whether these metal transporters would increase the cytotoxicity of gallium in gallium nitrate-resistant CCRF-CEM cells. Transferrin, in increasing concentrations, enhanced the cytotoxicity of gallium nitrate. One mg/ml transferrin decreased the 50% inhibitory concentration of gallium nitrate from 1650 to 75 micrometer in gallium-resistant cells and from 190 to 150 micrometer in gallium-sensitive cells. Transferrin also enhanced the cytotoxicity of gallium even at drug concentrations that were not growth inhibitory. The gallium chelate Ga-PIH inhibited the growth of both gallium nitrate-resistant and -sensitive cells. Fifty micrometer Ga-PIH inhibited cellular proliferation by 50%, whereas similar concentrations of PIH or gallium nitrate were not growth inhibitory. However, because higher concentrations of PIH also inhibited cell growth, the cytotoxicity of Ga-PIH was greater than PIH only at concentrations of <100 micrometer. Cross-titration experiments demonstrated that the cytotoxicity of PIH was partially reversed by gallium nitrate, whereas the cytotoxicity of gallium nitrate was enhanced by PIH. Our studies suggest that Ga-PIH warrants further evaluation as a potential antineoplastic agent. Because transferrin increases the cytotoxicity of gallium nitrate in transferrin receptor-bearing, gallium nitrate-resistant cells, future clinical trials of this drug should incorporate the development of strategies to increase plasma transferrin levels.

Effect of hydroxyurea on cellular iron metabolism in human leukemic CCRF-CEM cells: changes in iron uptake and the regulation of transferrin receptor and ferritin gene expression following inhibition of DNA synthesis.

Hydroxyurea inhibits cellular proliferation through action on ribonucleotide reductase, an iron-dependent enzyme responsible for the synthesis of deoxyribonucleotides. Whereas previous investigations have examined the interaction of hydroxyurea with this enzyme, the action of hydroxyurea on other aspects of iron metabolism has not been studied in detail. In our study, incubation of CCRF-CEM cells with hydroxyurea resulted in an inhibition of ribonucleotide reductase activity/DNA synthesis within 4 h and produced a parallel decrease in the uptake of iron by cells. In contrast, iron uptake by hydroxyurea-resistant CCRF-CEM cells was not inhibited by hydroxyurea. After 6 h, hydroxyurea produced an increase in the activity of the iron-regulatory protein, a cytoplasmic mRNA-binding protein responsible for regulating the translation of transferrin receptor and ferritin mRNAs. After 24 h, hydroxyurea-treated cells displayed a 1.5-fold increase in transferrin receptor mRNA and protein and a significant decrease in ferritin levels. The hydroxyurea-induced increase in transferrin receptor was abrogated by transferrin-iron. In contrast to hydroxyurea, inhibition of DNA synthesis by 1-beta-D-arabinofuranosylcytosine produced a decrease in transferrin receptor expression. Our studies suggest that iron uptake by CCRF-CEM cells is closely linked to ribonucleotide reductase activity rather than to transferrin receptor number. Inhibition of ribonucleotide reductase/DNA synthesis by hydroxyurea results in a decrease in iron uptake by cells and an increase in the activity of the iron-regulatory protein, which, in turn, is responsible for the hydroxyurea-induced increase in transferrin receptor and decrease in ferritin synthesis.

Induction of apoptosis by iron deprivation in human leukemic CCRF-CEM cells.

It is known that iron is essential for cell growth and viability and that iron deprivation results in an inhibition in the synthesis of deoxyribonucleotides. However, steps leading to eventual cell death during iron deprivation are not fully understood. In the present study, we report that cellular iron-deficiency produced by exposure of human leukemic CCRF-CEM cells to gallium or the iron chelator deferoxamine (DFX) resulted in the inhibition of cell growth, condensation of chromatin, and the formation of DNA fragments (DNA-ladder), findings that are characteristic of apoptotic cell death. These effects of gallium and DFX were detected after a 48-hour incubation with cells and could be prevented by ferric ammonium citrate (FAC). Iron-deprivation produced a small increase in the endogenous expression of bcl-2 protein. Our studies provide additional information regarding the mechanism of cytotoxicity of gallium and DFX, and suggest, for the first time, a role for iron in the suppression of apoptotic cell death.

Synergistic inhibition of T-lymphoblastic leukemic CCRF-CEM cell growth by gallium and recombinant human alpha-interferon through action on cellular iron uptake.

Gallium, a metal with clinical antineoplastic activity, is known to inhibit cellular iron uptake and iron-dependent DNA synthesis. Little information exists regarding the efficacy of gallium in combination with other agents. Since alpha-interferon (IFN-alpha) can modulate the action of certain chemotherapeutic drugs, we examined its influence on the growth inhibitory effects of gallium in CCRF-CEM cells. IFN-alpha and gallium as single agents had only minimal to moderate antiproliferative effects. In combination, however, both drugs synergistically inhibited cell growth, causing cell death accompanied by DNA fragmentation. At lower concentrations (120 microM), gallium inhibited cellular iron uptake but did not increase transferrin receptor expression, nor did it block cellular proliferation. The addition of IFN-alpha to this concentration of gallium significantly increased the gallium-induced block of iron uptake, resulting in an increase in transferrin receptors and an inhibition of cell growth. In contrast, IFN-alpha did not enhance the effects of the iron chelator deferoxamine on iron uptake or cell growth. Our studies suggest that gallium and IFN-alpha synergistically inhibit DNA synthesis through a mechanism that includes inhibition of cellular iron uptake and depletion of intracellular iron below the critical level needed to maintain DNA synthesis.