Combination iron depletion therapy.

Iron (Fe) depletion with anti-transferrin (Tf) receptor monoclonal antibodies (MAbs), Fe chelators, or gallium (Ga) salts inhibits in vitro and in vivo growth of tumor cells. The present studies examined the cytotoxic effects of an IgA anti-human Tf receptor MAb, 42/6, combined with parabactin, a powerful Fe chelator, or Ga nitrate. Parabactin inhibited in vitro growth of human hematopoietic and solid tumor cells, and the rank order of their sensitivities to the Fe chelator was identical to their relative sensitivity to MAb 42/6 as demonstrated in previous studies. When the most parabactin and MAb 42/6-sensitive (HL60 leukemia) and -resistant (KB carcinoma) cells were incubated with various concentrations of parabactin, cell killing was time and dose dependent over the first 24 hours. Little additional cytotoxicity occurred, however, when cells were exposed to parabactin for 48 hours. HL60 cells were slightly more sensitive than KB cells to parabactin cytotoxicity. Addition of optimally effective concentrations of anti-Tf receptor MAb 42/6 to parabactin increased cytotoxicity to HL60 cells over a narrow parabactin dose range but had little effect on cytotoxicity to KB cells. Cell cycle analysis of cells treated with parabactin for 24 hours showed that doses causing variable cytotoxicity increased the percentage of cells in S phase, but higher parabactin concentrations consistently arrested cells in G1 phase or at the G1/S interface. MAb 42/6 also increased toxicity of parabactin to granulocyte/macrophage colony-stimulating factors and normal marrow granulocyte/macrophage progenitors. When HL60 or KB cells were treated with MAb 42/6 combined with Ga nitrate, MAb 42/6 increased cytotoxicity of Ga for HL60 cells but had little or no effect on Ga cytotoxicity to KB cells. In contrast, MAb 42/6 had minimal effects on cytotoxicity of the ribonucleotide reductase inhibitor, isoquinaldehyde thiosemicarbazone, to either HL60 or KB cells. Both hematopoietic and solid tumors were killed by Fe depletion, but the present studies suggested that hematopoietic cells are more sensitive than solid tumor cells to cytotoxic effects of Fe depletion. Combined Fe depletion therapy by the use of MAb 42/6 with an Fe chelator or Ga salt increased toxicity to MAb 42/6-sensitive cells, such as HL60, but was not more effective against MAb 42/6-resistant solid tumor cells. Combination Fe depletion therapy of hematopoietic cell tumors merits evaluation in experimental in vivo tumor systems.

Effects of anti-epidermal growth factor (EGF) receptor antibodies and an anti-EGF receptor recombinant-ricin A chain immunoconjugate on growth of human cells.

The effects of antiepidermal growth factor (EGF) receptor monoclonal antibodies (MAbs) 528 and 225 and a 528-ricin A conjugate on the growth of normal and malignant human cells were tested in vitro. Malignant human cell lines with EGF receptor numbers ranging from 0 to 4 X 10(5) receptors/cell, human fetal fibroblasts, and normal marrow granulocyte/macrophage progenitors (CFU-gm) showed no effect when grown with 10(-12) M to 10(-7) M MAb 225 or 528. MAbs 225 and 528 and EGF also had no effect on the ability of marrow stromal cells to maintain CFU-gm viability in long-term marrow cultures. Reversible growth inhibition of A431 epidermoid and MDA-468 breast carcinoma cells with 2 and 3 X 10(6) EGF receptors/cell, respectively, was observed with both antibodies and with 10(-8) M EGF. In contrast, an immunoconjugate prepared with MAb 528 and recombinant ricin A chain (528-rRA) showed dose-dependent killing over a concentration range of 10(-12) M to 10(-8) M against cells with greater than or equal to 1.2 X 10(5) EGF receptors/cell [concentration that causes 50% inhibition of growth (IC50) values, approximately 10(-12) M to 10(-10) M]. Human fetal fibroblasts (5.6 X 10(4) EGF receptors/cell), melanoma cells without detectable EGF receptors, and human CFU-gm showed IC50 values of greater than 10(-8) M. Killing of KB epidermoid carcinoma cells and 547 ovarian carcinoma cells with 4 and 1.2 X 10(5) EGF receptors/cell by 10(-10) or 10(-11) M 528-rRA was time dependent, but cytotoxicity to 547 cells was not complete even with 48 hours of immunotoxin treatment. Cytotoxicity of 528-rRA was not enhanced by chloroquine or verapamil. In vitro, anti-EGF receptor MAbs cause reversible antiproliferative effects only against malignant cell lines with amplified EGF receptor expression. In contrast, 528-rRA shows potent, specific toxicity to cells with greater than 50,000 EGF receptors/cell. However, kinetics of cell killing with 528-rRA are protracted, suggesting that prolonged exposure may be required for in vivo antitumor effects.

Gamma-interferon modulates human monocyte/macrophage transferrin receptor expression.

Although circulating human monocytes do not express transferrin (Tf) receptors, cultured adherent blood cells display high-affinity Tf binding sites. In the present studies, effects of various cytokines and biologically active proteins on human monocyte/macrophage Tf receptors were investigated. After culture, Tf receptor expression by adherent blood cells was time dependent and plateaued by 7 days. The addition of interleukin-1 (IL-1), alpha-interferon (alpha-IFN), granulocyte/macrophage-colony stimulating factor (GM-CSF), or human IgG to macrophages cultured for 4 days did not alter Tf receptor expression. Fe-saturated, human Tf caused a significant, dose-dependent decrease in receptor expression. At a dose of 100 U/mL, gamma-interferon (gamma-IFN) significantly increased Tf receptor expression by macrophages cultured for 4 (230% +/- 51% of control) or 7 days (150% +/- 22%). Scatchard analyses showed increased binding sites but no change in receptor affinity. Northern and slot blot analysis of cellular mRNA from macrophages cultured for 4 to 7 days and exposed to gamma-IFN showed a two- to fivefold increase in Tf receptor mRNA, but less than or equal to 30% increase in beta-actin mRNA. Ferritin content of gamma-IFN-treated macrophages was 47% to 63% of control cells. Net uptake of 59Fe from Tf by gamma-IFN-treated cells was 10% to 17% of control, but uptake of radiolabeled Tf was comparable. When macrophages were labeled with 59Fe and then exposed to gamma-IFN, cell-associated Fe was reduced by 43%, indicating that gamma-IFN caused macrophage Fe release. gamma-IFN specifically modulates Tf receptor display by inducing Fe release and reducing cellular Fe content. Regulation of Tf receptor expression in macrophages is controlled by cellular Fe content and is thus similar to regulatory mechanisms in dividing cells.

Use of nude mouse xenografts as preclinical screens. Characterization of xenograft-derived melanoma cell lines.

Cell lines derived from human melanoma xenografts were characterized for surface markers, karyotype abnormalities, and in vitro drug sensitivity. Xenografts were established using metastatic explants from untreated patients and passaged in nude mice. Cell lines were readily established from melanoma xenografts, and formed colonies when plated in semisolid media. The lines expressed human melanoma-associated and other surface antigens, human lactate dehydrogenase (LDH) isoenzymes, and contained only human chromosomes. They failed to express murine histocompatibility determinants and were negative for murine viruses by mouse antibody production assay. Karyotypes showed abnormalities of chromosomes 3, 6, and 7 similar to other melanomas. In vitro chemosensitivity profiles were compared using cell line and xenograft colony-forming assays. Values were similar for the original xenografts and their cell lines. Xenograft-derived human melanoma lines resemble other melanoma cell lines and primary melanomas with respect to surface antigens and karyotype abnormalities, and are appropriate models for studying in vitro drug sensitivity. When used as a model for transition from solid tumor to cell line, these studies suggest cell lines closely mirror in vitro chemosensitivities of parent tumor cells. However, occasional, unpredictable changes in sensitivity to some drugs occurs during this transition.

Effects of monoclonal anti-transferrin receptor antibodies on in vitro growth of human solid tumor cells.

To determine whether anti-transferrin (Tf) receptor monoclonal antibodies might be useful in treatment of human solid tumors, in vitro effects of immunoglobulin A (42/6) and immunoglobulin G (B3/25) anti-Tf receptor antibodies on human solid tumor growth were examined. In colony and liquid cultures containing 10% serum, B3/25 did not inhibit growth of melanoma or ovarian carcinoma cell lines. 42/6 caused modest dose-dependent inhibition in colony cultures (maximum inhibition approximately 50%), and slowed growth of melanoma, ovarian carcinoma and epidermoid carcinoma cells in liquid culture. Inhibition was more pronounced in low (1%) serum, and was abrogated by 200 micrograms/ml iron-saturated Tf or 50 microM ferric nitriloacetate. All cells displayed high affinity Tf receptors (4-20 X 10(4)/cell). Cells grown in 1% serum and epidermoid carcinoma cells displayed more receptors, and susceptibility to 42/6 inhibition appeared related to higher receptor number. After culture with anti-Tf receptor antibodies, solid tumor cells showed a 57-93% reduction in surface Tf-binding sites. Tf uptake by cells grown for 24 h in B3/25 was approximately 50% of control, but was reduced to less than 10% of control with 42/6. Immunofluorescence staining of melanoma and HL60 promyelocytic leukemia cells suggested greater heterogeneity of Tf receptor display on melanoma than on leukemia cells. Previous studies showed 42/6 completely blocked blood cell Tf internalization and is a potent inhibitor of hemopoietic cell growth. In contrast, in solid tumor cells, inhibition of Tf uptake and growth inhibition are subtotal. Solid tumor resistance to 42/6 may be due in part to greater heterogeneity of Tf receptor display by proliferating cells. However, responses to iron-saturated Tf and ferric nitriloacetate in the presence of 42/6 also differ in hemopoietic and solid tumor cells, suggesting possible differences in Tf processing or iron growth requirements.

Use of nude mouse xenografts as preclinical drug screens. Further studies on in vitro growth of xenograft tumor colony-forming cells.

Previous studies suggested tumor colony-forming cells (CFC) grown from xenografts might be useful as a preclinical, in vitro drug screen. To further evaluate this possibility, eight melanoma and six ovarian carcinoma xenografts were established from untreated patients and tested for in vitro CFC growth. For each tumor, linear relationships between cells plated and colony (30 cells or greater than 75 micron diameter) and cluster (10-30 cells or 50-75 micron) growth were observed. All eight melanomas grew sufficient colonies (greater than or equal to 30) for in vitro drug assessment, although four required hypoxic (pO2 = 40) incubation to reliably attain this level of growth. Only one in six of the ovary xenografts consistently grew enough colonies, and growth was not significantly improved by hypoxic incubation, or addition of luteinizing hormone, follicle-stimulating hormone, or steroid hormones. Cloning efficiencies (colonies + clusters/cells plated) for tumors demonstrating adequate growth ranged from 0.01% to 0.3%. For most tumors, no direct relationship was observed between characteristics of xenograft tumors (size) or their resulting cell suspensions (viabilities, cell yield) and CFC growth. Cell suspensions were incubated with a 3 log concentration of nine established chemotherapeutic agents. Resulting dose-effect curves were linear and showed no plateaus of drug effect. Analyzing 447 in vitro drug trials on six melanomas and one ovarian carcinoma, interexperiment variability was high. Cell lines were established from three xenografts using a low concentration of fetal bovine serum (1%), and also examined for in vitro drug sensitivity. Using both liquid culture isotope incorporation and a colony-forming assay, drug sensitivity profiles for the cell lines were nearly identical to those for parent xenograft CFC. However, assays performed using the cell lines were more reproducible than those using xenograft tissue. The authors conclude that tumor CFC can be reliably grown from melanoma xenografts, but in vitro drug assays using these xenografts are poorly reproducible. The xenografts are a resource for establishing cell lines, and drug assays performed using these lines are highly reproducible. Similarities in drug sensitivity profiles for parent xenograft CFC and derived cell lines suggest that, despite poor reproducibility, repetitive assays using melanoma CFC accurately reflect some properties of cells which sustain tumor cell growth.

Transferrin modulation of colony stimulating factor elaboration by adherent blood and bone marrow cells.

When added to cultured normal, adherent blood and marrow cells at concentrations of 25-100 micrograms/ml (3 X 10(-7) to 1.3 X 10(-6) M), human transferrin enhanced colony-stimulating factor (CSF) elaboration. Fe saturated and relatively unsaturated Tf were equally effective in increasing CSF release, but soluble ferric nitriloacetate was ineffective. Dose-dependent increases in CSF release by adherent blood and marrow cells occurred in serum-free media and the continuous presence of Tf was necessary for this effect. Using a monoclonal anti-Tf receptor antibody, normal blood mononuclear cells contained no detectable Tf receptor positive cells. However, after 5 d culture in serum-free medium, Tf receptor positive cells were identified among normal adherent blood cells, and the per cent receptor positive cells increased with Tf concentration. We conclude Tf modulates CSF production from normal, adherent blood and marrow cells in vitro. These findings indicate a possible role for Tf in intramedullary regulation of normal granulopoiesis.

Heterogeneity of Ia antigen expression by proliferating nonlymphocytic leukemia blast cells.

In vitro systems were used to detect Ia-like antigens on proliferating normal myeloid and acute nonlymphocytic leukemia (ANLL) blast cells. Incubation of normal bone marrow cells with a monoclonal anti-Ia antibody and complement resulted in toxicity for both granulocyte/macrophage progenitors (CFU-GM) (toxicity 79%-100%) and cells proliferating in liquid culture in response to placenta-conditioned medium colony-stimulating factor (CSF) or medium conditioned by normal, phytohemagglutinin (PHA)-stimulated mononuclear cells. In contrast, effects of anti-Ia antibody and complement on blast colony-forming cells and 3H-TdR incorporation in liquid culture from eight patients with ANLL were variable. Colony growth with CSF after treatment was 0% to 91% of control growth and did not correlate with display of Ia-like antigens. Survival of ANLL cells growing in liquid cultures was even more variable after anti-Ia+ complement treatment (28%-227% of control). The presence of Ia-like antigens did not distinguish ANLL cells responding to PHA-conditioned medium from those responding to CSF in either colony or liquid culture. Dose-response curves for ANLL cells in liquid culture were similar before and after treatment with anti-Ia+ complement. In contrast to normal myeloid precursor cells, which show uniform display of Ia-like antigens, display of Ia antigen by proliferating leukemia cells is highly variable from patient to patient. Anti-Ia reagents such as this one would not be effective in treating ANLL marrow for autologous transplantation.

Effects of anti-transferrin receptor antibodies on growth of normal and malignant myeloid cells.

The effects of three monoclonal antibodies (B3/25, 43/31, and 42/6) reactive with human transferrin (Tf) receptors on growth of normal and malignant myeloid cells were examined using in vitro culture techniques. When added directly to cultures, all three antibodies caused dose-dependent inhibition of normal granulocyte/macrophage progenitor (CFU-GM) growth. Monoclonal antibody 42/6 was by far the most potent of the three, with an ID50 of less than 5 micrograms/ml. Identical effects were seen on CFU-GM from three patients with chronic myelogenous leukemia. Growth of colonies from two myeloid leukemia cells lines (KG-I, HL60) was also inhibited by all three antibodies, and these cells were generally more sensitive than normal CFU-GM. Blast colony-forming cells from three patients with acute non-lymphocytic leukemia were relatively resistant to the antibodies, and CFU-GM from a patient with myeloid metaplasia were resistant (ID50 greater than 50 micrograms/ml) to 42/6. In liquid culture, growth of the leukemic cell lines was inhibited by saturating concentrations of the three antibodies, although in both liquid and colony culture recovery was seen even after exposure to antibody for periods of up to 72 h. Analysis of the cell-cycle status of these cells showed that the antibodies did not cause accumulation of cells in any particular phase of the cell cycle. Addition to cultures of large quantities of human Tf failed to reverse the inhibitory effects of the antibodies. Competitive binding studies on the leukemia cell lines showed that only 42/6 inhibited binding of Tf to its receptor, although all three antibodies inhibited cell growth. Addition of Fe chelate (as ferric nitriloacetic acid, FeNTA) failed to reverse the inhibitory effects of the antibodies on CFU-GM and HL60 cells, but had variable effects on KG-I cell growth. FeNTA fully reversed inhibitory effects of 42/6 on KG-I cells. We conclude that monoclonal antibodies to Tf receptors can inhibit growth of both normal and malignant myeloid cells. Overall, no selectivity for malignant vs normal cells is apparent, although malignant cells from one individual were more sensitive to colony inhibition by 43/31 monoclonal antibody than normal CFU-GM.

Regulation of de novo purine synthesis in human bone marrow mononuclear cells by hypoxanthine.

In previous studies from this laboratory, human bone marrow hypoxanthine concentrations were found to average 7.1 microM, three times higher than plasma hypoxanthine concentrations measured simultaneously. To assess the significance of this finding, the relationship between hypoxanthine concentration and the rate of purine nucleotide synthesis by the de novo pathway was studied in normal human bone marrow mononuclear cells and in the human promyelocytic cell line, HL-60, in vitro. Utilizing a [14C]formate incorporation technique, rates of total cellular de novo purine synthesis as well as rates of de novo adenine, de novo guanine, and thymine synthesis and incorporation into RNA and DNA were measured as a function of hypoxanthine concentration. In normal human marrow cells, the rate of total de novo purine synthesis declined by 81%, while the rate of de novo adenine and de novo guanine synthesis and incorporation into macromolecules declined by 89 and 75%, respectively, when media hypoxanthine was increased from 0 to 10 microM. Similar results were seen in the HL-60 cell line. In contrast, rates of thymine synthesis and incorporation into DNA as well as overall rates of RNA and DNA synthesis did not change with varying media hypoxanthine concentrations. In addition, hypoxanthine salvage and incorporation into RNA and DNA was shown to progressively increase with increasing media hypoxanthine concentrations. These results indicate that physiologic concentrations of hypoxanthine are sufficient to regulate the rate of de novo purine synthesis in human bone marrow in vivo.

Glucocorticoid-mediated inhibition of ornithine decarboxylyase activity in S49 lymphoma cells.

Incubation of wild-type S49 lymphoma cells with glucocorticoids, such as dexamethasone and hydrocortisone, inhibits the activity of ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17), the rate-limiting enzyme in the pathway of polyamine biosynthesis. The kinetics of this inhibition are more rapid than the glucocorticoid-mediated growth arrest in the G1 phase of the cell cycle or in glucocorticoid-mediated cytolysis of these cells. The inhibition of ornithine decarboxylase activity by corticosteroids is specific for steroids of the glucocorticoid class. Results obtained with variant S49 cells having lesions in the pathways of glucocorticoid or cyclic AMP action indicate that cytoplasmic glucocorticoid receptors, as well as nuclear transfer of steroid--receptor complexes, are required for the inhibition of ornithine decarboxylase activity but that this inhibition does not require hormonal activation of adenylate cyclase or cyclic AMP-dependent protein kinase. Because glucocorticoid-mediated inhibition of ornithine decarboxylase occurs when cellular protein synthesis has decreased less than 20%, this inhibition may represent a specific glucocorticoid-mediated deinduction of ornithine decarboxylase in S49 cells. Inhibition of ornithine decarboxylase activity may offer a useful marker for suppression of growth and cell cycle progression in these and other lymphoma cells.

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities of S49 lymphoma cells by agents increasing cyclic AMP.

We have examined the regulation of two key enzymes that control polyamine biosynthesis-L-ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) - by agents increasing cAMP in S49 lymphoma cells. Incubation of wild type S49 cells with beta-adrenergic agonists (terbutaline or isoproterenol) inhibited ODC and SAMDC activities rapidly (less than 2 hr). more quickly than these agents arrested the cells in the G1 phase of the cell cycle. The beta-adrenergic antagonist propranolol blocked inhibition of ODC activity produced by isoproterenol, but only if added simultaneously or less than 4 hr after the agonist. Incubation of wild type S49 cells with cholera toxin or PGE1 also inhibited ODC activity. Decreases in ODC activity produced by beta-adrenergic agonists, cholera toxin, PGE1 or dibutyryl cAMP were all enhanced by the phosphodiesterase inhibitor Ro 20-1724. Results of studies of ODC and SAMDC activity in S49 variants having lesions in the pathway of cAMP generation and action were as follows: kin- cells (which lack cAMP-dependent protein kinase activity) showed no inhibition of ODC by any agent; AC- cells (which have absent nucleotide coupling units in their adenylate cyclase system) only demonstrated inhibition in response to dibutyryl cAMP; UNC cells (which have deficient coupling of hormone receptors and adenylate cyclase) only demonstrated inhibition in response to dibutyryl cAMP and cholera toxin, and beta-depleted cells (which have a decreased number of beta-adrenergic receptors) responded as did wild type cells except for absent response to isoproterenol. We conclude that inhibition of ODC and SAMDC activity in S49 cells is an early response to agents that increase cAMP and that this action occurs via the "classical" pathways of activation of adenylate cyclase and protein kinase. These results in S49 cells contrast with evidence in other systems in which cAMP has been suggested to enhance polyamine biosynthesis, perhaps through alternative mechanisms.