Inhibition of malignant cell growth by 311, a novel iron chelator of the pyridoxal isonicotinoyl hydrazone class: effect on the R2 subunit of ribonucleotide reductase.

The key roles of iron and iron proteins in cell proliferation make them potential targets for cancer therapy. However, clinical trials directed toward perturbation of tumor iron homeostasis by iron chelation have been limited to the use of deferoxamine (DFO). There is thus a need to develop agents with greater efficacy. In the present study, we investigated the mechanism of cytotoxicity of 311 (2-hydroxy-1-naphthylaldehyde benzoyl hydrazone), a novel iron chelator of the pyridoxal isonicotinoyl class. We found that 311 inhibited the growth of CCRF-CEM cells in a time- and concentration-dependent fashion with an IC(50) that was approximately 20-fold lower than that of DFO. 311 also inhibited the growth of breast, bladder, and head and neck cancer cell lines. Using electron spin resonance (ESR) spectroscopy analysis, we found that a 12-h exposure of CCRF-CEM cells to 311 inhibited the tyrosyl radical ESR signal of the R2 subunit of ribonucleotide reductase. However, overproduction of the R2 subunit in hydroxyurea-resistant CCRF-CEM cells was associated with a decrease in sensitivity of cells to 311 but not to DFO. Our studies show that 311 is a more potent cytotoxic agent than DFO, with activity against both hematopoietic and nonhematopoietic cell lines regardless of their p53 status. Furthermore, the ESR studies suggest that inhibition of the R2 subunit of ribonucleotide reductase is at least one mechanism by which 311 blocks cell proliferation.

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.

The anti-malarial artesunate is also active against cancer.

Artesunate (ART) is a semi-synthetic derivative of artemisinin, the active principle of the Chinese herb Artemisia annua. ART reveals remarkable activity against otherwise multidrug-resistant Plasmodium falciparum and P. vivax malaria. ART has now been analyzed for its anti-cancer activity against 55 cell lines of the Developmental Therapeutics Program of the National Cancer Institute, USA. ART was most active against leukemia and colon cancer cell lines (mean GI50 values: 1.11+/-0.56 microM and 2.13+/-0.74 microM , respectively). Non-small cell lung cancer cell lines showed the highest mean GI50 value (25.62+/-14.95 microM) indicating the lowest sensitivity towards ART in this test panel. Intermediate GI50 values were obtained for melanomas, breast, ovarian, prostate, CNS, and renal cancer cell lines. Importantly, a comparison of ART's cytotoxicity with those of other standard cytostatic drugs showed that ART was active in molar ranges comparable to those of established anti-tumor drugs. Furthermore, we tested CEM leukemia sub-lines resistant to either doxorubicin, vincristine, methotrexate, or hydroxyurea which do not belong to the N.C.I. screening panel. None of these drug-resistant cell lines showed cross resistance to ART. To gain insight into the molecular mechanisms of ART's cytotoxicity, we used a panel of isogenic Saccaromyces cerevisiae strains with defined genetic mutations in DNA repair, DNA checkpoint and cell proliferation genes. A yeast strain with a defective mitosis regulating BUB3 gene showed increased ART sensitivity and another strain with a defective proliferation-regulating CLN2 gene showed increased ART resistance over the wild-type strain, wt644. None of the other DNA repair or DNA check-point deficient isogenic strains were different from the wild-type. These results and the known low toxicity of ART are clues that ART may be a promising novel candidate for cancer chemotherapy.

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.

Interaction of gallium nitrate with other inhibitors of ribonucleotide reductase: effects on the proliferation of human leukemic cells.

Ribonucleotide reductase, a key enzyme in deoxyribonucleotide synthesis, is an important target for cancer chemotherapy. Drugs that inhibit its individual components may act synergistically to block DNA synthesis. Prior work has established that gallium inhibits the R2 subunit of ribonucleotide reductase. We show that gallium acts synergistically with the ribonucleotide reductase inhibitors gemcitabine and hydroxyurea to inhibit the proliferation of CCRF-CEM cells. In contrast, combinations of gallium with the ribonucleotide reductase inhibitors amidox, didox, or trimidox produced antagonistic effects on cell growth. Spectroscopy analysis revealed that as a result of their metal-binding properties, amidox, didox and trimidox formed complexes with gallium, thus negating potential synergistic actions. Our results have important implications in the design of clinical trials using these ribonucleotide reductase inhibitors in combination.

Gallium-pyridoxal isonicotinoyl hydrazone (Ga-PIH), a novel cytotoxic gallium complex. A comparative study with gallium nitrate.

The anti-proliferative activity of gallium-pyridoxal isonicotinoyl hydrazone (Ga-PIH), a novel gallium complex was compared with that of gallium nitrate, a known anti-tumor agent. At 50 microM, Ga-PIH inhibited CCRF-CEM cell growth by 45% compared to < 10% inhibition with gallium nitrate or PIH. The IC50s for Ga-PIH, gallium nitrate and PIH were 60, 84 and 68 microM respectively. The addition of exogenous iron as transferrin-iron to the culture medium reversed the cytotoxicity of gallium nitrate and PIH in a dose-dependent manner but had only minor effects on the cytotoxicity of Ga-PIH. The effect of these compounds on cellular iron uptake was measured since prior studies have shown that gallium perturbs iron transport into cells. Fifty micromolar Ga-PIH, gallium nitrate or PIH inhibited the cellular uptake of 59Fe-transferrin over 24 h by 65%, 32%, and 78% respectively. Although all three compounds inhibited iron uptake, only Ga-PIH produced a significant upregulation of cellular transferrin receptors. Since the cytotoxicity of Ga-PIH appears to be less influenced by extracellular iron and cellular transferrin receptor expression, it may have potential as an antineoplastic agent and should be further evaluated in animal tumor models.

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 continuous-infusion gallium nitrate and hydroxyurea in combination for the treatment of refractory non-Hodgkin's lymphoma.

Based on preclinical studies demonstrating synergy between gallium and hydroxyurea, we evaluated the efficacy and toxicity of continuous intravenous gallium nitrate in combination with oral hydroxyurea in patients with refractory non-Hodgkin's lymphoma. Fourteen patients, median age 64 years (range 53-89), with stage III or IV low- or intermediate-grade lymphoma were treated with gallium nitrate and hydroxyurea in combination for 7 days at four different dose levels: (a) gallium nitrate, 200 mg/m2/day; hydroxyurea, 500 mg/day; (b) gallium nitrate, 250 mg/m2/day; hydroxyurea, 1,000 mg/day; (c) gallium nitrate, 300 mg/m2/day; hydroxyurea, 1,000 mg/day; and (d) gallium nitrate, 350 mg/m2/day, hydroxyurea, 1,000 mg/day. All patients had progressive disease and had been heavily pretreated. Six of 14 patients had objective tumor regression following treatment (one complete response, one near-complete response, and four partial responses) with a median duration of response of 7 weeks (range 3-38 weeks). An additional four patients had minor responses. Responses occurred at all dose levels and in both low- and intermediate-grade histologic subtypes. The predominant toxicities encountered were anemia and reversible nephrotoxicity. Combination gallium nitrate and hydroxyurea has significant activity in lymphoma and is well tolerated even by elderly patients. Because of the lack of cross-resistance to other drugs and the potential synergistic antineoplastic activity, gallium nitrate and hydroxyurea should be further evaluated in combination with other chemotherapeutic agents.

Effect of gallium nitrate therapy on Ga-67 scintigraphic detection of lymphoma: case report.

Ga-67 and abdominal CT scans of a 72-year-old woman who had malignant lymphoma before, during, and after gallium nitrate/hydroxyurea combination therapy are presented. Disappearance of Ga-67 uptake by the tumor during this treatment despite continuing CT evidence of disease and reappearance of Ga-67 scan abnormalities after cessation of therapy suggests that caution should be exercised when interpreting results of Ga-67 scintigraphy for the detection of tumor viability during gallium nitrate/hydroxyurea therapy.

Modulation of in vitro and in vivo T-cell responses by transferrin-gallium and gallium nitrate.

Gallium is a group IIIa metal that has efficacy in the therapy of malignant disorders such as lymphoma and urothelial tract tumors. Preclinical studies also indicate a role for gallium in autoimmune disorders, suggesting that gallium is able to modulate T-cell immune reactivity. The purpose of this study was to examine the in vitro and in vivo immunomodulatory action of gallium on T-cell function. Since gallium binds to transferrin in vivo, in vitro studies evaluated the effect of transferrin-gallium (Tf-Ga) on human T cells. Tf-Ga inhibited the mitogen-induced proliferative response of peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion. Alloantigen-induced proliferation was also potently suppressed when evaluated in a mixed lymphocyte culture assay. Tf-Ga affected a significant reduction in the density of IL-2 receptors on activated T cells and a slight reduction in the number of CD3+/CD25+ T cells in PHA-stimulated cultures. Neither secretion of interleukin-2 (IL-2) nor the induction of IL-2-stimulated lymphokine-activated killer activity, however, was inhibited by Tf-Ga. Tf-Ga produced significant upregulation of the transferrin receptor (CD71) in T cells as determined by flow cytometric analysis and northern blot assay, but did not affect the percentage of CD3+/ CD71+ T cells after mitogen stimulation. To assess the in vivo effects of gallium on alloreactive T cells, we evaluated the immunosuppressive effect of gallium in a murine model of graft-versus-host disease (GVHD). Administration of gallium significantly prolonged survival in mice undergoing severe GVHD, suggesting that gallium can ameliorate GVH reactivity. Collectively, these data demonstrate that, at clinically achievable concentrations, Tf-Ga potently inhibits T-cell activation and that this immunosuppressive property of gallium may be of adjunctive therapeutic value in the management of disorders characterized by the presence of autoreactive or alloreactive T-cell populations.

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.

Gallium nitrate delays the progression of microscopic disease in a human medulloblastoma murine model.

The goal of adjuvant chemotherapy is to treat postoperative microscopic disease in the hope of preventing tumor recurrence and/or metastasis. Since the introduction of chemotherapeutic agents, the disease-free survival of children with medulloblastoma has improved only modestly. Therefore, there is a need to develop and investigate new chemotherapeutic agents for this malignancy. Gallium nitrate has demonstrated significant antineoplastic activity toward human medulloblastoma in vitro and in vivo and may prove to be an optimal chemotherapeutic agent in treating medulloblastoma microscopic disease. The present study consisted of injecting medulloblastoma Daoy intradermally into both flanks of nude mice. A 15-day 50-mg/kg/day regimen was implemented the day after tumor inoculation. All treated and control mice received saline hyperhydration during the treatment period. The interval between tumor cell inoculation and first measurable tumor detection, tumor occurrence, growth rate, and size were recorded. Results indicated that gallium nitrate significantly prolonged the interval between tumor cell inoculation and measurable tumor detection.

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.

Prevention of gallium toxicity by hyperhydration in treatment of medulloblastoma.

In vitro and in vivo studies have established gallium nitrate as an effective chemotherapeutic agent against human medulloblastoma. In vitro, gallium nitrate reduced cell proliferation and DNA synthesis of medulloblastoma Daoy. Gallium inhibits the availability of 59Fe to ribonucleotide reductase and has a direct effect on the enzyme itself. In vivo, gallium demonstrated similar effects on the medulloblastoma Daoy cell line in nude mice. Tumor growth rate and actual size were decreased; however, severe nephrotoxicity and mortality were observed. In our study, intradermal injections of medulloblastoma Daoy cells were given to nude mice and then tumors were allowed to grow. Tumor-bearing mice received a 15-day gallium (50 mg/kg/day) regimen, 20-day rest, 7-day gallium (66.5 mg/kg/day) dose escalation regimen beginning when tumor size exceeded 8-10 mm in diameter. All treated and control mice received saline hyperhydration during both treatment sessions. Our study resulted in the prevention of severe toxicity and an inhibition of tumor growth. No toxicity occurred with gallium nitrate at 50 mg/kg/day. Severe morbidity and mortality were observed at the higher gallium dose level (66.5 mg/kg/day), suggesting that the 50 mg/kg/day dose is the appropriate level when investigating gallium nitrate as a chemotherapy agent in nude mice.

Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 and lymphoid CCRF-CEM leukaemic cells.

Gallium binds to the iron transport protein transferrin (Tf), is incorporated into cells through transferrin receptors (TfR) and inhibits iron-dependent DNA synthesis. Since cellular TfR expression is tightly regulated by the availability of iron, we investigated the effects of transferrin-gallium (Tf-Ga) on TfR mRNA levels in myeloid HL60 and lymphoid CCRF-CEM cells. In HL60 cells, Tf-Ga increased TfR mRNA levels in a dose-dependent fashion. This increase in TfR mRNA was blocked by Tf-Fe and by cycloheximide. Analysis of the rate of mRNA decay in the presence of actinomycin D revealed that the half-life of TfR mRNA was increased in HL60 cells incubated with Tf-Ga. The rate of transcription of TfR mRNA was not increased by Tf-Ga. In contrast with HL60 cells, CCRF-CEM cells displayed a decrease in the level of TfR mRNA after incubation with Tf-Ga. Tf-Ga inhibited iron uptake in both HL60 and CCRF-CEM cells but increased the level of TfR mRNA only in HL60 cells, suggesting that the Tf-Ga induction of TfR mRNA was not solely due to inhibition of cellular iron uptake. At growth-inhibitory concentrations, Tf-Ga increased the TfR mRNA level in HL60 cells but decreased it in CCRF-CEM cells. Our studies suggest that in HL60 cells, gallium regulates TfR expression at the post-transcriptional level by mechanisms which require de novo protein synthesis and involve interaction with iron. The divergent effects of Tf-Ga on TfR mRNA in myeloid HL60 and lymphoid CCRF-CEM cells suggest that differences exist in the regulation of TfR expression between these two cell types.

Effect of gallium on the tyrosyl radical of the iron-dependent M2 subunit of ribonucleotide reductase.

Gallium, a pharmacologically important metal which resembles iron, was shown in previous studies to inhibit ribonucleotide reductase. To better understand its mechanism of action, we have examined the interaction of gallium with the iron-dependent M2 subunit of ribonucleotide reductase. In its active form, M2 contains an iron center and a tyrosyl free radical which is detectable by ESR spectroscopy. In the present study, cytoplasmic extracts prepared from murine leukemic L1210 cells after an 18-hr incubation with 960 microM gallium nitrate displayed a > 60% inhibition in their M2 tyrosyl radical ESR signal. However, this signal was restored within 15 min to levels greater than that of controls by the addition of increasing concentrations of ferrous ammonium sulfate. Gallium citrate added directly to cytoplasmic extracts from control cells also decreased the tyrosyl radical signal, an effect which could be reversed by iron. Immunoblot analysis revealed that incubation with gallium did not diminish the amount of M2 protein in cells, thus indicating that the decrease in the tyrosyl radical signal was not due to a decrease in cellular M2 content. In immunoprecipitation studies of 59Fe-labeled M2, gallium displaced 55-60% of the 59Fe incorporated into M2. Our studies suggest that gallium displaces iron from the M2 subunit of ribonucleotide reductase, resulting in a loss of the tyrosyl radical and an accumulation of inactive M2 within the cell.