Cytokines in chronic inflammatory arthritis. II. Granulocyte-macrophage colony-stimulating factor in rheumatoid synovial effusions.

A liquid culture technique was used to study 23 synovial fluids (SF) (21 from inflammatory joint diseases and 2 noninflammatory SF) and supernatants of two cultured rheumatoid arthritis (RA) synovial tissues for colony-stimulating factor (CSF). The proliferative responses of human peripheral blood macrophage-depleted non-T cells treated with synovial fluids, supernatants of synovial tissue explants, and recombinant granulocyte-macrophage (rGM)-CSF were compared. Aggregates of cells that formed in long-term cultures (15 d) were similar for each applied agent and consisted of macrophages, eosinophils, and large blasts. Tritiated thymidine incorporation was proportional to the concentration of rGM-CSF and was accompanied by an increase in number and size of cellular aggregates formed in the cultures. CSF activity was observed in inflammatory SF, with tritiated thymidine uptake of 3,501 +/- 1,140 cpm in the presence of RA samples (n = 15) compared to 1,985 +/- 628 for non-RA inflammatory SF (n = 7) (P less than 0.05) and 583 +/- 525 for medium (n = 6) (P less than 0.01). The proliferative response to RA SF was often more apparent when the samples were diluted, because at higher concentrations the RA SF was inhibitory. Two RA SF were fractionated by Sephadex G100 column chromatography; low levels of CSF activity were detected in fractions corresponding to Mr of 70-100 kD, but the major CSF activity was found in the 20-24-kD fractions. A polyclonal rabbit anti-GM-CSF antibody eliminated the stimulating activity from both rGM-CSF and RA SF. Finally, a specific RIA identified significant levels of GM-CSF (40-140 U/ml) in the culture supernatants of 3 additional RA synovial tissues. These data document the local production of GM-CSF in rheumatoid synovitis and are the first description of this cytokine at a site of disease activity.

Role of transferrin, Fe, and transferrin receptors in myeloid leukemia cell growth. Studies with an antitransferrin receptor monoclonal antibody.

In previous studies, antitransferrin receptor antibody 42/6 inhibited growth of normal granulocyte/macrophage progenitors and some malignant myeloid cells. In these studies, leukemia cell lines cultured without serum and fresh leukemia cells were used to investigate the roles of Fe, transferrin receptors, and transferrin in leukemia cell growth, and mechanisms of 42/6 inhibition and resistance. HL60 and KG-1 leukemia cells grown in serum-free medium were inhibited by 42/6. In contrast to results in fetal calf serum (FCS), soluble Fe (ferric nitriloacetate) reversed 42/6 growth inhibition of serum-free HL60 cells. When HL60 cells were adapted for growth in serum-free, transferrin-free medium, they became refractory to 42/6 growth inhibition. By using radiolabeled transferrin and 42/6, HL60 cells cultured in FCS and transferrin displayed similar quantities of transferrin receptors (29,000-30,000/cell) and similar Kd's (3.8-4.9 X 10(-9) M). Cells grown in transferrin-free medium showed a similar Kd (3.1 X 10(-9) M), but fewer transferrin binding sites (5,000/cell). Transferrin-independent cells contained a log higher concentration of intracellular ferritin. For both FCS and serum-free HL60 cells, calculated affinities for 42/6 were lower (5.7-10.0 X 10(-9) M), but the number of binding sites was three- to fourfold higher. To investigate further the relationship between receptor display and antibody inhibition in proliferating normal and malignant myeloid cells, simultaneous immunofluorescence was used to determine the cell cycle status of transferrin receptor-positive cells. Malignant cells in S + G2/M displayed approximately 50% of the amount of transferrin receptors detected in normal dividing colony-stimulating factor-stimulated marrow cells. Receptor display by dividing cells from two patients with acute nonlymphocytic leukemia was variable. When HL60 cells were exposed to dimethyl sulfoxide, transferrin receptor display decreased, and 42/6 growth inhibition was abrogated or greatly diminished. The presence of 42/6 did not prevent dimethyl sulfoxide-induced HL60 differentiation in serum-containing or serum-free cultures. We conclude that human leukemia cells require Fe for growth and that 42/6 inhibits transferrin-dependent cells by Fe deprivation. Some dividing normal and differentiating malignant cells display reduced transferrin receptors, and can also escape antibody inhibition. The increased ferritin levels and decreased transferrin receptors in transferrin-independent HL60 cells confirm the inverse relationship between cell ferritin content and transferrin receptor display. These studies indicate a critical role for Fe in leukemia cell growth and possible roles in cellular differentiation.

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.

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.

Preclinical evaluation of illudins as anticancer agents: basis for selective cytotoxicity.

Illudins are potent natural products derived from Omphalotus illudens and related fungi. The chemical structure of illudins differs from that of other conventional chemotherapeutic agents. While illudins are toxic to most tumor cells after prolonged exposure (greater than or equal to 48 hr), with shorter exposure times (less than or equal to 2 hr), they show selective toxicity for human myelocytic leukemia and epidermoid, lung, ovarian, and breast carcinoma cells of various species of origin. The apparent histologic specificity of illudin S toxicity is based on an energy-dependent transport mechanism present in sensitive cells, but absent in cells relatively resistant to illudin S. For human myeloid leukemia HL60 cells, the Michaelis constant was 4.2 microM and the maximum velocity was 12.2 pmol/minute per 10 million cells or 730 pmol/hour per 10 million cells. The energy-dependent transport mechanism was detected in other mammalian tumor cells.

Effects of combined antigrowth factor receptor treatment on in vitro growth of multiple myeloma.

BACKGROUND: Although passive serotherapy for cancer with monoclonal antibodies is an attractive concept, it has unfortunately had limited efficacy in clinical trials. An alternative approach to passive serotherapy is targeting cell surface growth factor receptors with monoclonal antibodies. With some limitations, anti-growth factor receptor antibodies can limit cell growth by blocking stimulatory or trophic growth factor receptors and by marshaling in vivo antitumor immune responses. PURPOSE: The purpose of our study was to determine the extent to which anti-interleukin-6 (IL-6) and anti-transferrin (Tf) receptor antibodies, when used individually or combined, could limit myeloma cell growth. METHODS: The four myeloma cell lines studied varied in IL-6 responses from factor independence (myeloma cell lines 8226 and U266) to strict factor dependence (OCI-My4 myeloma cells and human acute myelogenous leukemia [AML] cell line UCSD/AML1). IL-6 RNA was detected using reverse transcriptase-polymerase chain reaction. IL-6 protein was detected in U266 supernatant by growth stimulation of UCSD/AML1 cells and by enzyme-linked immunosorbent assay. For cell growth assays, cell lines were plated with various concentrations of IL-6 and anti-receptor antibodies and [3H]thymidine uptake determined after 3 days. Cells were grown in varying concentrations of IgG1 monoclonal anti-Tf receptor antibodies E2.3 and A27.15 or antibodies PM1, AUK 146-15, AUK 64-7, or AUK 12-20 to the human IL-6 receptor-alpha protein. Tf and IL-6 receptors were detected by immunofluorescence staining. RESULTS: Using short-term proliferation assays, anti-Tf receptors and anti-IL-6 antibodies caused dose-dependent growth inhibition of varying degrees, and, in one of three cell lines, a combination of anti-Tf and anti-IL-6 antibodies showed supra-additive growth inhibition. IL-6-independent cells were inhibited by anti-Tf receptor antibodies, while IL-6-dependent cells were resistant to these antibodies but sensitive to anti-IL-6 receptor. Factor-dependent myeloma cells exposed to either anti-Tf or anti-IL-6 receptor antibodies for 48 hours lost colony-forming capability. A combination of anti-Tf and anti-IL-6 antibodies increased elimination of colony-forming cells at 24 hours. CONCLUSIONS: Anti-receptor antibodies have distinct patterns of myeloma cell growth inhibition and inhibit in vitro growth of factor-dependent myeloma cells. Combinations of anti-growth factor receptor antibodies also increase toxicity for IL-6-dependent myeloma colony-forming units.

Antiproliferative and antitumor activity of the 2-cyanoaziridine compound imexon on tumor cell lines and fresh tumor cells in vitro.

BACKGROUND: Imexon, a 2-cyanoaziridine, is therapeutic and reverses lymphadenopathy and splenomegaly in the LP-BM5 murine retrovirus-induced immunodeficiency disease (murine AIDS). It can restore chemotherapy-induced immunosuppression. Imexon reduced the incidence of lymphoma in severe combined immune deficient mice inoculated with human lymphocytes. PURPOSE: To determine its antitumor activity, we screened imexon against fresh human tumor cells and tumor cell lines. To determine the time-concentration relationships of its cytotoxicity, we studied the effects of imexon on macromolecular synthesis and on the cell cycle. METHODS: Imexon was incubated at 1-200 micrograms/mL with various tumor cell lines, mitogen-stimulated peripheral blood lymphocytes, and fresh tumor cells. Cell survival, macromolecular synthesis, and cell cycle progression were studied. RESULTS: The concentration of imexon that caused 50% inhibition of growth was under 10 micrograms/mL for lymphocytes stimulated with mitogens. It was about 3-10 micrograms/mL for B-cell lymphomas and both multi-drug-resistant and -sensitive myeloma cell lines. Imexon inhibited four of seven fresh lymphoma and 11 of 16 fresh myeloma biopsy specimens to less than 40% of the control. A 1-hour exposure of lymphoma cells to 50-100 microgram/mL followed by removal of drug by washing the cells and continuing culture resulted in greater than 95% inhibition during the next 48-72 hours. Imexon selectively inhibited protein synthesis during the first 24-48 hours of exposure of lymphoma and myeloma cells. Cells exposed to inhibitory concentrations of imexon were blocked in cell cycle progression. CONCLUSION: Imexon may be a potentially useful agent in the treatment of malignant disease, particularly lymphoid malignancies, and should be explored further.

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.

Mechanisms of growth inhibition by anti-transferrin receptor monoclonal antibodies.

In previous studies, an immunoglobulin A, anti-transferrin receptor antibody (42/6) inhibited growth of a variety of normal and malignant human hemopoietic cells. To determine its mechanism of growth inhibition, we compared effects of 42/6 and B3/25, an immunoglobulin G anti-transferrin receptor antibody which does not inhibit lymphocyte growth, on transferrin (TF) binding and uptake. As in previous studies, affinity constants of TF and anti-TF receptor antibodies for human TF receptors at 4 degrees C were similar, but the number of calculated binding sites was higher for the antibodies. Antibody B3/25 did not inhibit TF binding at either 4 degrees C or 37 degrees C. At 4 degrees C, antibody 42/6 inhibited TF binding to normal, mitogen-stimulated mononuclear cells. However, TF did not inhibit 42/6 binding, suggesting 42/6 inhibited TF binding by noncompetitive, possibly steric, mechanisms. When cells were simultaneously exposed to labeled TF and unlabeled anti-TF receptor antibodies at 37 degrees C, 42/6 inhibited TF binding only slightly. Initial uptake of antibodies and TF at 37 degrees C was rapid, but when mononuclear cells or HL60 cells were cultured with either 42/6 or B3/25 for 2 days, TF binding and immunoreactive TF receptor sites decreased. However, TF bound to cells cultured with B3/25 continued to enter the cell, whereas cells cultured with 42/6 would no longer take up bound TF. Studies using HL60 cells grown with soluble iron in lieu of TF showed that changes in TF binding sites and TF uptake were not secondary to growth inhibition. We conclude that incubation with both inhibitory (42/6) and noninhibitory (B3/25) anti-TF receptor antibodies results in decreased TF binding sites. However, exposure to 42/6 also inhibits TF uptake and causes growth inhibition by iron deprivation. Monoclonal antibodies to receptors transporting critical nutrient molecules, such as iron, may inhibit cell growth by blocking ligand access to the cell's interior.

Response of human myeloid leukemia cells to various sources of colony-stimulating activity and phytohemagglutinin-conditioned medium.

The response of human myeloid leukemia cells to various sources of colony-stimulating activity (CSA) and media conditioned by phytohemagglutinin-stimulated mononuclear cells (PHA-LCM) was investigated in liquid and colony culture. PHA-LCM, placenta-conditioned medium, GCT cell line-conditioned medium, leukocyte-conditioned medium, and partially purified CSA for human and murine cells were tested for ability to support growth of granulocyte-macrophage colonies from adherent cell-depleted human bone marrow. This activity was correlated with ability to support leukemia colony growth in methylcellulose, and [3H]thymidine incorporation in liquid culture by normal bone marrow cells, leukemia cells, and the KG-1 myeloid leukemia cell line. For normal cells, growth and liquid culture responses were highly correlated for various sources of CSA (r = 0.92), and addition of data using PHA-LCM changed results only slightly (r = 0.89). [3H]thymidine incorporation by leukemia cells from patients without a prior history of a myeloproliferative disorder was also highly correlated with normal CSA (r = 0.97) for sources other than PHA-LCM. Responses of leukemia blasts and KG-1 cells in liquid culture to PHA-LCM appeared in excess of its CSA for normal cells. Colony growth by leukemia cells was not clearly correlated with either liquid culture activity for leukemia cells or CSA for normal cells. PHA-LCM was also not statistically superior to placenta-conditioned medium as stimulus for leukemia colony growth, but was superior to placenta-conditioned medium for some patients. Differentiation in culture did not appear to depend on CSA source. We conclude that normal myeloid cells respond to CSA in a highly correlated fashion in both colony and liquid cultures. The majority of myeloid leukemia cells respond to either PHA-LCM or CSA, but the ability of PHA-LCM to support leukemia cell growth is greater than its CSA content. The possibility exists that overlapping populations responsive to CSA and to PHA-LCM are present simultaneously in patients with myeloid leukemia.

In vitro sensitivity to steroid hormones and cytotoxic agents of normal and malignant lymphocyte colony-forming cells.

In vitro assays were used to assess the sensitivity of normal T-cells and malignant, chronic lymphocytic leukemia (CLL) lymphocyte colony-forming cells (CFC) to a panel of cytotoxic drugs and steroid hormones. Normal T-CFC were remarkably resistant to hydrocortisone, progesterone, estradiol, and testosterone at concentrations less than or equal to 10(-5) M. Variable inhibition was seen at concentrations of 10(-4) M, and prior exposure to phytohemagglutinin increased sensitivity only to sex steroid hormones. In contrast to T-CFC, which showed little variation in patterns of steroid hormone inhibition in vitro, CLL-CFC from individual patients displayed widely varying sensitivity to all hormones tested; 50% inhibitory dose varied by as much as 2 logs. T-CFC were fairly resistant to a 1-hr exposure to achievable concentrations of 1-beta-D-arabinofuranosyl-cytosine, 5-fluorouracil, chlorambucil, melphalan, cisplatin, methotrexate, Adriamycin, and bleomycin. Prior exposure to phytohemagglutinin resulted in increased sensitivity only to low concentrations of Adriamycin, a phenomenon that appeared related to prior or concurrent lectin exposure and not to changes in cell cycle status. CLL-CFC showed variable sensitivity to Adriamycin and cisplatin, and concurrent exposure to lectin and Adriamycin did not increase sensitivity to that drug. CLL cells displayed much greater sensitivity to a 1-hr exposure to antimetabolites and bleomycin than to continuous exposure to the same drugs. In contrast to normal T-CFC, CLL-CFC exposed to methotrexate were not "rescued" by subsequent culture in media and fetal bovine serum. Incubation of T-CFC or CLL-CFC with melphalan and a source of protein (fetal bovine serum or bovine serum albumin) resulted in decreased cell kill. Differences in in vitro sensitivity of normal and malignant lymphocyte CFC to steroids and cytotoxic agents can be demonstrated using these culture systems. CLL-CFC showed variable sensitivity to hydrocortisone, and much greater sensitivity to antimetabolites than normal T-CFC. Differences in conditions of drug exposure, such as concurrent exposure to lectin or inclusion of protein, may alter the in vitro sensitivity of lymphocyte CFC to some drugs.

In vitro growth and drug sensitivity of tumor colony-forming units from human tumor xenografts.

To investigate the feasibility of using tissue obtained from human tumor xenografts for in vitro screening of antineoplastic agents, we grew human tumor colony-forming units (CFU) in semisold agar from xenografts serially passaged in nude mice. Growth of human tumor CFU was accomplished from nine xenografts representing five different histological tumor types (ovarian carcinoma, adenocarcinoma of the colon, malignant melanoma, epidermoid carcinoma of the lung, and malignant astrocytoma). Cloning efficiency ranged from 0.04 to 0.1% and showed significant variability both between tumor types and between individual animals bearing the same type of xenograft. A high percentage of tumor CFU was in S phase [47 +/- 20% (S.D.)] as determined by the thymidine "suicide" technique. The number of tumor CFU observed increased linearly with increasing numbers of cells plated. In vitro drug sensitivity of the tumor CFU was assessed to Adriamycin, cis-platinum, and melphalan. The patterns of drug sensitivity were found to be reproducible and stable over a period of 9 months. Drug sensitivity curves to Adriamycin for five xenografts representing four tumor types showed complex patterns with plateau portions similar to those described for tumor CFU from primary tumors. The rank order of sensitivity of the tumors was compared to that of normal granulocyte-macrophage progenitors and, with the exception of the melanomas, was found to correlate well with clinical experience (order of sensitivity = colon less than ovary less than bone marrow). Growth of human tumor CFU from xenografts represents a reproducible and stable means for the study of the biology of tumor CFU and has potential applications as a means for screening new anticancer agents.

Assessment of ligand effects in intracellular trafficking of ricin A chain using anti-ricin hybridomas.

Intracellular ricin and immunotoxin trafficking has been difficult to study as only one to two cytosolic ricin A chain (RTA) molecules are sufficient to cause cell death. Previous studies (R.J. Youle and M. Colombatti, J. Biol. Chem., 262: 4676-4882, 1987) using anti-ricin hybridomas identified the secretory pre-Golgi as a critical site for RTA intoxication. We used ricin and RTA immunotoxins constructed with transferrin (TF) or anti-murine TF receptor antibody (RI7/217) to compare patterns of cytotoxicity and intracellular trafficking in anti-ricin hybridomas. Anti-RTA and anti-ricin B chain (RTB) hybridomas bound similar amounts of ricin and secreted comparable amounts of anti-ricin immunoglobulin. Anti-RTA hybridomas were 50- to 500-fold more resistant to ricin than nonsecretory and anti-RTB hybridomas, defining a ricin-resistant phenotype. All hybridomas expressed similar levels of surface TF receptors. RTA immunotoxins were constructed using human TF or RI7/217 and a disulfide linker. In protein synthesis inhibition assays, ricin-resistant hybridomas were manyfold more resistant to RI7/217-RTA than were ricin-sensitive hybridomas. In contrast, all hybridomas were equally sensitive to TF-RTA. Monensin increased ricin cytotoxicity minimally against all hybridomas, but dramatically increased RI7/217-RTA cytotoxicity in ricin-resistant and ricin-sensitive hybridomas in a way that abrogated the ricin-resistant phenotype. In contrast, monensin increased TF-RTA cytotoxicity equally in all hybridomas. Ammonium chloride had little effect on ricin or RI7/217-RTA cytotoxicity, but increased TF-RTA cytotoxicity against all hybridomas. Taken together, these results suggest that RTA molecules mediating cytotoxicity pass through an anti-RTA antibody-containing pre-Golgi compartment when bound to RTB or RI7/217, but not when bound to TF. Monensin abrogates the ricin-resistant phenotype when RTA is linked to RI7/217, but not RTB. This suggests that monensin alters RI7/217-RTA processing proximal to the pre-Golgi and that passage through the pre-Golgi may not be necessary for translocation of RTA to the cytoplasm. Ammonium chloride alters toxin cytotoxicity only when RTA is linked to TF, suggesting that only TF trafficks RTA through an acid-sensitive compartment prior to cytoplasmic translocation. With the addition of potentiating agents, each toxin studied showed a unique cytotoxicity profile against the anti-ricin hybridomas, demonstrating a dominant role of the cell binding ligand in intracellular toxin trafficking.

Effects of transforming growth factor beta 1 on growth and apoptosis of human acute myelogenous leukemia cells.

Because limited studies examined effects of transforming growth factor (TGF) beta 1 on growth of human acute myelogenous leukemia (AML) cells, we used factor-dependent and primary AML cells to assess TGF-beta 1 effects on human AML cell growth. OCI-AML1 cells were growth inhibited by TGF-beta 1 regardless of which growth factor was used as a stimulus. In contrast, AML-193 cells were resistant to TGF-beta 1 when grown with or without growth factors. UCSD/AML1 cells were sensitive to TGF-beta 1 inhibition when grown with most cytokines but were relatively resistant to TGF-beta 1 in the presence of macrophage colony-stimulating factor (M-CSF). Although cells grown from 5 of 6 AML patients were inhibited by TGF-beta 1, cells from 1 AML patient were growth stimulated by TGF-beta 1 in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, or mast cell growth factor (kit ligand). Thus, 3 growth patterns with TGF-beta 1 were observed: (a) sensitivity to growth inhibition; (b) resistance; and (c) factor-dependent resistance. Further studies showed that AML-193 and UCSD/AML1 cells expressed type II TGF-beta 1 receptors and that ability of TGF-beta 1 to decrease GM-CSF receptors did not correlate with growth inhibition. AML-193 cells and UCSD/AML1 cells grown with M-CSF could be propagated in 1 ng/ml TGF-beta 1, but UCSD/AML1 cells grown with GM-CSF and TGF-beta 1 died. Morphology and agarose gel analysis of DNA showed UCSD/AML1 cells underwent apoptosis when grown with GM-CSF and TGF-beta 1 but not with M-CSF and TGF-beta 1. Similar studies of OCI-AML1 cells showed that TGF-beta 1 induced apoptosis of cells grown in 5637 bladder cell-conditioned medium or GM-CSF. These studies indicate that human AML cells exhibit heterogeneous growth responses to TGF-beta 1 and that some effects of TGF-beta 1 on myeloid cells occur through programmed cell death.

Expression of functional epidermal growth factor receptors in a human hematopoietic cell line.

To test the feasibility of using the human epidermal growth factor receptor (EGFR) as a model for growth factor receptor action in human hematopoietic cells, we infected Burkitt lymphoma cells (Namalwa) with a recombinant amphotrophic retrovirus containing a thymidine kinase promoter-driven human EGFR complementary DNA and the neomycin resistance gene. Neomycin-resistant cells expressing surface EGFR were selected by cell sorting using anti-EGFR monoclonal antibody 225. The selected cells expressed a Mr 170,000 protein immunoprecipitated by monoclonal antibody 225 and apparently identical to EGFR from A431 carcinoma cells. Infected Namalwa cells expressed 42,000 epidermal growth factor (EGF) binding sites/cell, and Scatchard analysis showed two affinities (Kd approximately 5 nM and approximately 0.5 nM). EGFR autophosphorylation was detected using antiphosphotyrosine antibodies after 5 min exposure to EGF. EGF binding induced rapid EGFR internalization (t1/2 = 9 min) and mobilization of transferrin receptors to the cell surface within 1 min. In fetal bovine serum-containing and serum-free cultures, EGF did not stimulate Namalwa cell proliferation. However, in the presence of 1.25% dimethyl sulfoxide (DMSO), EGF caused a dose-dependent increase in DNA synthesis. DMSO induced a cell cycle block in G1, which was partially reversed by EGF. DMSO induced changes in some B-cell markers suggesting cellular differentiation and increased surface EGF receptor number. Cells grown in DMSO and EGF were established as an EGF-dependent cell line for greater than 12 weeks, whereas cells grown in DMSO without EGF died within 1-2 weeks. Namalwa cells expressing EGFR demonstrated more rapid tumor growth in athymic mice. These studies demonstrate expression of functional EGFR mediating early biochemical and growth responses in a human hematopoietic cell, and indicate that EGFR can be used as an effective model in human hematopoietic cells. Results using DMSO are consistent with studies in other human leukemia cells indicating that agents inducing differentiation can restore growth factor dependence in previously factor-independent leukemia cells.

Combinations of anti-transferrin receptor monoclonal antibodies inhibit human tumor cell growth in vitro and in vivo: evidence for synergistic antiproliferative effects.

Thirty-two murine monoclonal antibodies (MAbs) against the external domain of the human transferrin (Tf) receptor have been obtained using human Tf receptor glycoprotein produced in a baculovirus expression system as immunogen. The MAbs were tested separately, and in combination, for their ability to inhibit growth of CCRF-CEM leukemic T-cells in tissue culture. One MAb, D65.30, an IgG1, inhibited growth of CCRF-CEM cells as effectively as the anti-Tf receptor MAb 42/6, an IgA, previously found to have the highest antiproliferative activity in this assay. Some combinations of two or more MAbs inhibited the in vitro growth of CCRF-CEM much more effectively than single MAbs. Eleven IgG1 anti-Tf receptor MAbs, when combined individually with D65.30, increased the inhibition of CCRF-CEM growth from approximately 65% to greater than 90% in a 7-day growth assay. Similarly, many IgG MAbs in combination with 42/6 also inhibited CCRF-CEM growth by greater than 90%. The growth-inhibitory effects of certain combinations of MAbs were clearly synergistic, because either one or both MAbs tested separately were inactive. These pairs of MAbs also inhibited the growth of HL-60 and KG-1 leukemic cells by greater than 90% and partially inhibited the growth of K562 erythroleukemia and M21 melanoma cells, which are resistant to MAb 42/6. However, not all combinations of anti-Tf receptor MAbs were more effective; eight MAbs markedly antagonized the antiproliferative effects of D65.30, whereas 12 others had little or no effect. Preincubation of HL-60 cells with three different pairs of MAbs, D65.30 and A27.15, B3/25 and 42/6, and B3/25 and TR3A, inhibited subsequent colony formation by greater than 95%, demonstrating that their action is cytotoxic, not cytostatic. The antiproliferative activity of these pairs of MAbs correlates with their ability to block Tf-mediated 59Fe uptake and perturb Tf receptor expression. Treatment of nude mice bearing established s.c. CCRF-CEM tumors with a combination of MAbs D65.30 and A27.15 inhibited tumor growth and in some animals led to complete tumor regression. Each MAb administered separately was much less effective. We conclude that combinations of two or more anti-Tf receptor MAbs can interact synergistically to inhibit cell growth in vitro and tumor growth in vivo.

Preclinical evaluation of illudins as anticancer agents.

Illudins are low molecular weight natural products which were previously evaluated as anticancer drugs using rodent tumor models. In the present studies, we used in vitro cultures of human cancer cells to reevaluate their potential as anticancer agents. Using continuous exposure, Illudins S and M were cytotoxic to human leukemia cells at concentrations of 6-100 nM, but dihydroilludin M was 3 orders of magnitude less toxic, thus identifying a ketone site as a structural feature critical for cytotoxicity. Cytokinetic studies showed that illudin S caused a complete block at the G1-S phase interface of the cell cycle. Kinetics of inhibition of radiolabeled thymidine, uridine, and leucine incorporation suggested a primary effect on DNA synthesis. In colony and liquid culture assays, cell killing was time dependent but near maximal with a 2-h exposure. Myeloid and T-lymphocyte leukemia cells were most sensitive (50% inhibitory concentration, 6-11 nM), but B-cell leukemia/lymphoma, melanoma, and ovarian carcinoma cells were at least 10 times more resistant. Bone marrow granulocyte/macrophage progenitors showed intermediate sensitivity. Illudin S was equally effective against CEM T-lymphocyte leukemia cells expressing the multidrug resistance phenotype associated with Mr 180,000 glycoprotein and the parental cell line. CEM cells resistant to doxorubicin, epipodophyllotoxins, and 1-beta-D-arabinofuranosylcytosine showed only a 2-fold increased resistance to illudin S. Illudins are novel and potent cytotoxins which may be preferentially active against human myeloid and T-cell leukemias, including cells resistant to more conventional chemotherapeutic agents. The present studies illustrate the breadth of information which can be obtained on a new agent using present in vitro screening procedures and human cells.

Efficacy of Acylfulvene Illudin analogues against a metastatic lung carcinoma MV522 xenograft nonresponsive to traditional anticancer agents: retention of activity against various mdr phenotypes and unusual cytotoxicity against ERCC2 and ERCC3 DNA helicase-deficient cells.

Four second-generation Illudin analogues were synthesized and tested for antitumor activity using a metastatic lung carcinoma xenograft model resistant to conventional antitumor agents. One analogue, the parent illudofulvene-derivative called Acylfulvene, inhibited xenograft primary tumor growth and prolonged life span of tumor-bearing animals when administered i.p. or i.v. The efficacy of Acylfulvene exceeded that of mitomycin C, cisplatin, paclitaxol, the parent compound Illudin S, and an earlier analogue, dehydroilludin M. Promising features of this new analogue are: (a) the retention of in vitro activity against a variety of mdr tumor phenotypes including gp170+, gp150+, GSHTR-Pi, topoisomerase I, and topoisomerase II mutants; and (b) an apparent selective cytotoxicity toward cells deficient in either ERCC2 or ERCC3 DNA helicase activity.

Chromosome microdissection identifies cryptic sites of DNA sequence amplification in human ovarian carcinoma.

DNA sequence amplification contributes to the multistep process of carcinogenesis, and overexpression of amplified genes has been shown to contribute to the malignant phenotype. Cytogenetic analyses of human tumor cells, including ovarian malignancies, frequently show cytological evidence of DNA amplification in the form of double minutes and homogeneously staining regions. In this report, we have combined the techniques of chromosome microdissection and fluorescence in situ hybridization (P. S. Meltzer et al., Nat. Genet., 1: 24-28, 1992) to identify the composition and chromosomal origin of seven homogeneously staining regions from seven cases of ovarian cancer. Twelve specific chromosome band regions were identified as amplified including 11q, 12p, 16p, 19p, and 19q. These results provide important insights into the organization of amplified sequences within ovarian malignancies and add further to our recognition of regions likely to harbor genes important to the development or progression of ovarian cancer.

Failure of low-dose, total-body irradiation to augment combination chemotherapy in extensive-stage small cell carcinoma of the lung.

In a pilot study, 21 consecutive eligible patients with extensive-stage small cell carcinoma of the lung were scheduled for treatment with combination chemotherapy followed by total-body irradiation (TBI), prophylactic cranial irradiation, and consolidative chemotherapy. Induction chemotherapy consisted of VP16-213, vincristine, cyclophosphamide, and doxorubicin (VOCA). TBI was given as 100 rads in 10 fractions over 2 wk. Consolidation chemotherapy consisted of cyclophosphamide, methotrexate, and hexamethylmelamine (CMH). VOCA chemotherapy was well tolerated, with a 79% response rate in 19 evaluable patients. TBI was successfully given after four cycles of VOCA without excessive morbidity in 11 patients, although subsequent CMH chemotherapy in 8 patients has required dose reductions and some delays in therapy. Unfortunately, TBI did not increase the degree of response, and 2 patients relapsed during this therapy. Median survival in this study was 40-44 wk. One patient has survived 78 wk and remains in remission. TBI can be safely given following induction chemotherapy in extensive-stage small cell carcinoma of the lung, but it does not appear to add to the therapeutic benefit of combination chemotherapy alone.