Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium.

We have previously shown that human leukemic cells proliferate normally in serum-free media containing various transferrin forms, but the addition of transferrin-gallium leads to inhibition of cellular proliferation. Because gallium has therapeutic potential, the effects of transferrin-gallium on leukemic cell proliferation, transferrin receptor expression, and cellular iron utilization were studied. The cytotoxicity of gallium is considerably enhanced by its binding to transferrin and cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms. Exposure to transferrin-gallium leads to a marked increase in cell surface transferrin binding sites, but despite this, cellular 59Fe incorporation is inappropriately low. Although shunting of transferrin-gallium to another cellular compartment has not been ruled out, other studies suggest that transferrin-gallium impairs intracellular release of 59Fe from transferrin by interfering with processes responsible for intracellular acidification. These studies, taken together, demonstrate that inhibition of cellular iron incorporation by transferrin-gallium is a prerequisite for inhibition of cellular proliferation.

Regulation of transferrin receptor expression on human leukemic cells during proliferation and induction of differentiation. Effects of gallium and dimethylsulfoxide.

The association of transferrin receptor expression with cellular proliferation has been studied extensively, but a number of events have not been defined. We therefore assayed receptor on promyelocytic leukemia (HL-60) cells at early times after exposure to a stimulus for proliferation (subculture), as well as agents that either induce differentiation (dimethylsulfoxide [DMSO] ) or inhibit iron uptake (transferrin-gallium). Within 4 h after subculture, we found that a significant increase in total cellular immunoreactive receptor occurred that preceded by 8 h the increase in cell-surface transferrin binding. Automated fluorocytometric analysis of cells in an immunofluorescent assay indicated that increased surface receptor density appeared on cells in the S, G2, and M phases of the cell cycle. DMSO-treated cells proliferated at the same rate as untreated (control) cells for the first 72 h, but as early as 12 h after treatment transferrin receptor was significantly decreased (65% of control cells). Further decreases occurred at later time points until transferrin receptor was undetectable after 7 d, when proliferation had ceased, cells were arrested in G1 phase of the cell cycle, and myeloid differentiation occurred. After exposure to transferrin-gallium, proliferation ceased, but cells exhibited increased surface receptor and were arrested at S phase of the cell cycle without associated myeloid differentiation. We conclude that events preceding cell division provide the regulatory stimulus for the synthesis and subsequent appearance of the transferrin receptor on the cell surface. Additionally, decreased receptor expression may be important in causing cessation of proliferation and/or differentiation. Finally, the way in which gallium salts are currently being investigated as chemotherapeutic agents should be reevaluated in light of our findings concerning transferrin-gallium effects on cellular proliferation.

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.

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.

Uptake of gallium-67 by human leukemic cells: demonstration of transferrin receptor-dependent and transferrin-independent mechanisms.

We have studied the role of transferrin and the transferrin receptor in the uptake of 67Ga by the human leukemic cell line HL60. In the absence of transferrin, HL60 cells incorporated about 1% of the 67Ga dose over 6 h. The presence of transferrin increased cellular 67Ga uptake approximately 10-fold. Transferrin-mediated uptake of 67Ga was blocked by an anti-transferrin receptor monoclonal antibody, and decreases in the density of cellular transferrin receptors led to corresponding decreases in the transferrin-dependent uptake of 67Ga. Changes in the cellular ferritin content did not significantly influence the uptake of 67Ga by either transferrin-independent or transferrin-dependent pathways. Regardless of the mechanism of uptake, a significant amount of intracellular 67Ga was found to be associated with immunoprecipitable ferritin as well as with a free pool. This free intracellular 67Ga appeared to be kinetically active since cells released 67Ga back to the media over time. Our results demonstrate the existence of a dual mechanism for the cellular uptake of 67Ga and suggest that the preferential uptake of 67Ga by lymphomas is related to the high density of transferrin receptors known to be expressed by these tumors in vivo.

Role of the acidic receptosome in the uptake and retention of 67Ga by human leukemic HL60 cells.

The uptake of 67Ga by HL60 cells requires binding of 67Ga-transferrin (Tf) to cell surface Tf receptors. To further examine this process, we have studied early events in the cellular uptake of 67GaTf. Cell surface-bound 67GaTf and 59FeTf displayed similar kinetics during the first 10 min of uptake. Thereafter, approximately 10% of intracellular 67Ga was released by cells while 59Fe internalization continued to increase with time. In pulse-chase studies of 125I-Tf-67Ga uptake, internalized 125I-Tf, but not 67Ga, was chased out of cells by nonradioactive Tf-Ga. Exposure of cells to monensin, a carboxylic ionophore, during initial uptake decreased the internalization of both 125I-Tf and 67Ga. Exposure to monensin at a later time, after cells had incorporated 125I-Tf-67Ga or 59FeTf, caused an increase in the release of 67Ga and 59Fe with a decrease in the release of 125I-Tf. Ammonium chloride inhibited the internalization of both 67Ga and 59Fe. 67GaTf uptake by HL60 cells involves initial internalization into an acidic receptosome. This is followed by dissociation of 67Ga and Tf and subsequent trafficking of each to separate intracellular compartments. Disruption of this process by monensin results in the release of 67Ga from cells.

Interaction of gallium nitrate with fludarabine and iron chelators: effects on the proliferation of human leukemic HL60 cells.

Earlier studies have shown that transferrin-gallium inhibits cellular iron incorporation and blocks DNA synthesis by decreasing the activity of the iron-dependent M2 subunit of ribonucleotide reductase. We examined the growth-inhibitory effects of gallium nitrate in combination with clinically relevant inhibitors of ribonucleotide reductase fludarabine (an M1 subunit inhibitor), and iron chelators (M2 subunit inhibitors). Fludarabine and gallium nitrate in combination produced a significant increase in cell growth inhibition when compared with either agent alone; however, this effect was partially reversible up to 24 h and was best seen with continuous exposure of cells to both drugs. Incubation of cells with desferrioxamine and gallium nitrate resulted in reversal of gallium-induced growth inhibition. Incubation of cells with N,N'-bis(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid and gallium nitrate resulted in a slight increase in gallium-induced growth inhibition, with partial restoration of cell growth occurring only at a single high concentration of N,N'-bis(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid. Both chelators inhibited 67Ga uptake by cells and increased cell surface transferrin receptors. In contrast to the coincubation studies, sequential exposure of cells to desferrioxamine or N,N'-bis(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid and gallium nitrate resulted in a significant potentiation of the growth-inhibitory effects of gallium nitrate. Our studies show that cellular iron deprivation results in enhanced sensitivity of cells to gallium. Furthermore, the combined effects of fludarabine and gallium on cell growth may be of clinical relevance, since both agents are active against lymphoid cancers.

Modulation of lymphocyte proliferation and immunoglobulin production by transferrin-gallium.

Gallium resembles iron with respect to transferrin (Tf) binding and cellular uptake via Tf receptors. We have previously shown that transferrin-gallium (Tf-Ga) complexes interfere with the cellular incorporation of iron and inhibit the proliferation of HL60 cells. Since mitogen-stimulated peripheral blood lymphocytes express Tf receptors, we examined the effect of Tf-Ga on lymphocyte proliferation and on immunoglobulin synthesis by B-lymphocytes. Tf-Ga inhibited phytohemagglutinin, pokeweed mitogen, and tetanus toxoid-stimulated lymphocyte proliferation by greater than 50%, an effect which appeared to be cytostatic rather than cytotoxic. In cocultures of T-lymphocytes or CD4+ T-lymphocytes and B-lymphocytes, Tf-Ga also inhibited pokeweed mitogen-stimulated immunoglobulin production by 84 to 100%. Tf-Ga inhibited both T-independent Epstein Barr virus-stimulated B-lymphocyte proliferation and immunoglobulin production; however, these effects appeared to be independent of each other, since immunoglobulin production was inhibited by 75% by a concentration of Tf-Ga which did not uniformly inhibit proliferation. Tf-Ga is capable of targeting Tf receptor-bearing T- and B-lymphocytes and interfering with their proliferation and function. Such effects may be of relevance to patients being treated with this metal. The potential immunosuppressive activity of gallium warrants further investigation.

Development of drug resistance to gallium nitrate through modulation of cellular iron uptake.

We have shown that transferrin-gallium (Tf-Ga) blocks DNA synthesis through inhibition of cellular iron incorporation and a diminution in the activity of the iron-dependent M2 subunit of ribonucleotide reductase. To examine the mechanisms of drug resistance to gallium, we developed a subline of HL60 cells (R cells) which is 29-fold more resistant to growth inhibition by gallium nitrate than the parent line (S cells). R cells displayed a 2.5-fold increase in transferrin (Tf) receptor expression, without a change in receptor affinity for Tf. The uptake and release of 67Ga were similar for both S and R cells. The uptake of 59Fe-Tf by S cells was inhibited by gallium nitrate over 24-48 h of incubation. In contrast, 59Fe-Tf uptake by R cells, although initially inhibited by gallium nitrate at 24 h, was no longer inhibited at 48 h of incubation. 59FeCl3 uptake by R cells was significantly greater than that of S cells, regardless of the time in culture. Despite the increase in 59Fe uptake by R cells, the ferritin content of these cells was lower than that of S cells. The ribonucleotide reductase electron spin resonance signal of R cells was comparable to that of S cells. R cells were not cross-resistant to Adriamycin, vincristine, cis-platinum or hydroxyurea. Resistance to gallium nitrate in this subline of HL60 cells results primarily from the ability of cells to overcome the gallium-induced block in iron incorporation. In addition, intracellular iron in R cells appears to traffic preferentially to a non-ferritin compartment.

Inhibition of ribonucleotide reductase by gallium in murine leukemic L1210 cells.

Our previous studies of the mechanism of cell growth inhibition by gallium have suggested that the block in cellular iron uptake induced by transferrin-gallium results in an inhibition of the iron-dependent M2 subunit of ribonucleotide reductase. However, it is not known whether the inhibitory effect of gallium on ribonucleotide reductase is solely the result of limiting iron availability for enzyme activity or whether a direct effect of intracellular gallium on the enzyme is also involved. In the present study, utilizing a cell-free assay, we show that gallium nitrate directly inhibits CDP and ADP reductase activity. Inhibition of DNA synthesis by gallium nitrate thus appears to be due to a combination of a block in iron availability to ribonucleotide reductase and a direct inhibition of the enzyme by gallium.

Allogeneic bone marrow transplantation for relapsed and refractory lymphoma using genotypically HLA-identical and alternative donors.

Twenty-two patients, ages 16.6 to 43.9 years (median age, 30 years), with relapsed or refractory lymphoma were treated by allogeneic bone marrow transplantation after high-dose chemotherapy with or without total body irradiation (TBI). Seven patients had Hodgkin's disease, four had low-grade histology non-Hodgkin's lymphoma (NHL), seven had intermediate-grade NHL, and four had high-grade NHL. Of the 22 patients, 17 received T-cell (CD-3)-depleted marrow after intensive pretransplant chemoradiotherapy, and five received T-cell-replete grafts after chemotherapy-based preparative regimens. Five patients were transplanted from donors other than genotypically HLA-identical siblings: four from partially HLA-matched relatives, and one from a phenotypically HLA-identical unrelated donor. Acute graft-versus-host disease (GVHD) was less than or equal to grade II in all patients, and chronic GVHD was limited or absent in all but one patient. Of the 21 assessable patients, 17 (80.9%) achieved complete remissions. Death due to transplant-associated complications occurred in five patients, and five patients have relapsed. Thirteen patients are alive, and 12 are continuously relapse-free at a median follow-up of longer than 28 months (range, greater than 10 to greater than 58 months) from transplant. The cumulative probability of treatment failure from relapse or progression of lymphoma was 29% (95% confidence interval [CI], 12% to 51%), while the actuarial lymphoma-free (ie, event-free) survival plateau is 54.6% (95% CI, 34% to 76%). For young patients with advanced malignant lymphoma, allogeneic bone marrow transplantation appears superior to salvage chemotherapy for achievement of long-term, lymphoma-free survival and may be preferable to autologous bone marrow transplantation for selected patients.

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.

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.

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.

Transferrin receptor-mediated suppression of in vitro hematopoiesis by transferrin-gallium.

The expression of transferrin receptors on cells is felt to reflect iron requirements for proliferation or for hemoglobin production. We have recently shown that transferrin-gallium (Tf-Ga) complexes bind to cellular transferrin receptors and inhibit cellular iron incorporation. In this study, Tf-Ga in a dose-dependent manner inhibited the growth of erythroid (erythroid burst-forming units [BFU-E]-derived), granulocyte-macrophage (granulocyte-macrophage colony-forming units [CFU-GM]-derived) and mixed (mixed CFU [CFU-GEMM]-derived) hematopoietic colonies. Although major differences in the response of the different progenitor cells to Tf-Ga were not seen, CFU-GEMM-derived colonies appeared to be more sensitive to growth inhibition by Tf-Ga. The inhibitory effects on colony growth were reversible after 48 h of exposure of marrow cells to Tf-Ga, suggesting that the initial effects of Tf-Ga were mainly cytostatic and that continuous exposure of cells to Tf-Ga was required for maximal growth inhibition. Transferrin-iron (Tf-Fe) added to the Tf-Ga-containing cultures restored colony growth; however, this effect was best seen when Tf-Fe was added at day 0 of incubation. Tf-Fe added on days 3 or 7 failed to restore GEMM colonies and restored only a fraction of BFU-E and GM colonies. Tf-Ga appears to inhibit hematopoietic progenitor cell growth by interfering with cellular iron utilization during an early phase of progenitor cell proliferation. The use of Tf-Ga may allow further exploration of the role of iron and the Tf receptor in the regulation of hematopoietic progenitor cell growth.

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.

Familial leukemia and aplastic anemia associated with monosomy 7.

A kindred is described in which eight of 14 patients in one generation had acute nonlymphocytic leukemia or aplastic anemia either alone or terminating in acute nonlymphocytic leukemia. The proband and two siblings in one branch of this kindred presented with aplastic anemia, whereas acute nonlymphocytic leukemia was the presenting feature in the other two branches. Karyotypic evolution from a normal karyotype to monosomy 7 was demonstrated in the proband, and group C monosomy was seen in two other patients. The proband's serum sample inhibited in vitro growth of normal bone marrow colonies. The occurrence of hematologic disease in this kindred appears to be the result of a maternally transmitted trait, and persons younger than 30 years of age appear to have the highest risk of hematologic disease.

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.

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.

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.