Complete suppression of in vivo growth of human leukemia cells by specific immunotoxins: nude mouse models.

In this study, immunotoxins containing monoclonal anti-human T-cell leukemia antibodies are shown to be capable of completely suppressing the tumor growth of human T-cell leukemia cells in vivo without any overt undesirable toxicity. These immunotoxins were prepared by conjugating ricin A chain (RA) with our monoclonal antibodies, SN1 and SN2, directed specifically to the human T-cell leukemia cell surface antigens TALLA and GP37, respectively. We have shown that these monoclonal antibodies are highly specific for human T-cell leukemia cells and do not react with various normal cells including normal T and B cells, thymocytes, and bone marrow cells. Ascitic and solid human T-cell leukemia cell tumors were generated in nude mice. The ascitic tumor was generated by transplanting Ichikawa cells (a human T-cell leukemia cell line) i.p. into nude mice, whereas the solid tumor was generated by transplanting s.c. MOLT-4 cells (a human T-cell leukemia cell line) and x-irradiated human fibrosarcoma cells into x-irradiated nude mice. To investigate the efficacy of specific immunotoxins in suppressing the in vivo growth of the ascitic tumor, we divided 40 nude mice that were injected with Ichikawa cells into four groups. Each group of 10 mice was injected with one of the following mixtures: 40 micrograms of purified control mouse IgG [IgG1(kappa)] (group 1), 40 micrograms of control RA conjugate (group 2), 20 micrograms of purified SN1 antibody [IgG1(kappa)] and 20 micrograms of purified SN2 antibody [IgG1(kappa)] (group 3), or 20 micrograms of SN1-RA and 20 micrograms of SN2-RA (group 4). Mice in groups 1 and 2 formed large ascitic tumors, and died 5.8-7.0 weeks after the transplantation. Group 3 mice also formed large ascitic tumors and died 6.4-7.8 weeks after the transplantation. However, none of the mice in group 4 that were treated with SN1-RA and SN2-RA showed any signs of a tumor or undesirable toxic effects for the 20 weeks that they were followed after the transplantation; these mice were indistinguishable from healthy control nude mice that were not injected with Ichikawa cells. Treatment with SN1-RA plus SN2-RA completely suppressed solid tumor growth in 4 of 10 nude mice carrying solid tumors and partially suppressed the tumor growth in the remaining 6 nude mice. These results strongly suggest that SN1-RA and SN2-RA may be useful for clinical treatment.     

Proliferation and maturation of human leukemic cells in liquid culture: activity of human placenta conditioned medium and retinoic acid

Marrow or peripheral blood cells from 28 patients with acute myeloid leukemia (AML) or chronic myeloid leukemia in blastic crisis (CML-BC) were studied in both liquid and agar cultures. The proliferation and maturation of these cells were followed for 15-20 days in liquid culture with or without the addition of human placenta conditioned medium (HPCM) and/or retinoic acid (RA). In nine patients (group 1), cells underwent both proliferation and maturation, i.e., the percentage of peroxidase-positive cells (PO), phagocytic cells, and mature forms increased. For the remaining 19 patients (group 2), no proliferation was observed. However, 11 of these leukemic cell samples showed maturation (group 2A), while the eight others remained immature (group 2B). In agar culture, cell samples from group 1 showed cluster growth, group 2 no growth. Maturation without proliferation was observed for group-1 liquid cultures not containing HPCM and those containing HPCM and RA. The viability rapidly decreased in liquid cultures with only addition of RA. HPCM and RA showed no effect on group-2 cell cultures.     

Role of B7-1 in mediating an immune response to myeloid leukemia cells

A costimulatory signal from B7-1 (CD80) to its counter-receptor CD28 is required for T-cell activation. Many tumors, including most human leukemias, lack expression of B7-1, and this has been suggested to contribute to the failure of immune recognition of these diseases. A murine leukemia model system was developed to assess the potential role of B7-1 in the induction immunity to leukemia cells. The nonleukemic 32Dc13 myeloid cell line was transformed by transfection of the BCR/ABL gene, generating a subline (32Dp210/clone 26) that was leukemic and rapidly lethal to syngeneic, immunocompetent C3H/HeJ mice or T-cell- deficient nude mice. B7-1-modified leukemic cells remained lethal in nude mice, but caused only a transient, nonlethal leukemia in C3H/HeJ mice. After a single exposure to live, nonirradiated B7-1-modified leukemic cells, C3H/HeJ mice developed protective immunity against subsequent challenge with B7-1(-) leukemic cells. Further, hyperimmunization with B7-1(+) leukemic cells prolonged the survival of mice previously injected with a lethal number of B7-1(-) leukemic cells. These results indicate that myeloid leukemic cells may be attractive candidates for B7-1 gene transfer.     

Friend murine leukemia virus-immortalized myeloid cells are converted into tumorigenic cell lines by Abelson leukemia virus.

Friend murine leukemia virus (Fr-MuLV) is a replication-competent murine retrovirus that induces acute nonlymphocytic leukemias in NFS/n mice. Fr-MuLV disease is divided into two stages based on the ability of the leukemia cells to grow in culture and transplant into syngeneic mice. Hematopoietic cells taken from the early stage of disease after Fr-MuLV infection grow as immortal myeloid cell lines in the presence of WEHI-3 cell-conditioned medium (CM) or interleukin 3. These growth factor-dependent cell lines do not grow in culture in the absence of CM and do not form tumors in syngeneic animals. If these Fr-MuLV-infected cells are superinfected with Abelson murine leukemia virus (Ab-MuLV), they lose their dependence on WEHI-3 CM and proliferate in culture in the absence of exogenous growth factors. Concomitant with the loss of growth factor dependence in culture, the Ab-MuLV-infected cell lines become tumorigenic in syngeneic mice. This secondary level of transformation is Ab-MuLV specific. Fr-MuLV-immortalized myeloid cell lines superinfected with Harvey murine sarcoma virus (Ha-MuSV) or amphotropic virus remain dependent on WEHI-3 CM for growth in vitro and are not tumorigenic in vivo. Neither Ab-MuLV- nor Ha-MuSV-infected normal mouse myeloid cell cultures produce growth factor-independent or tumorigenic cell lines. We conclude that at least two genetic events are needed to convert a murine myeloid precursor into a tumorigenic cell line. The first event occurs in Fr-MuLV-infected mice, generating cells that are growth factor dependent but immortal in vitro. The second event, which can be accomplished by Ab-MuLV infection, converts these immortal myeloid precursors into growth factor-independent and tumorigenic cells.     

The role of interleukin-1 and granulocyte-macrophage colony-stimulating factor in the paracrine stimulation of an in vivo-derived murine myeloid leukemia

WEHI-274.3 is a cell line isolated from an in vivo-derived, murine myelomonocytic leukemia. Although the survival and growth of WEHI-274.3 cells in vitro is absolutely dependent on the addition of exogenous growth factors such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or colony-stimulating factor-1, when injected into syngeneic mice the cell line is tumorigenic. Sera from normal mice contain low levels of an activity that sustains survival of WEHI-274.3 but does not stimulate growth. In contrast, sera from mice bearing the WEHI-274.3 leukemia contained levels of CSF-1 and GM-CSF that stimulated the growth of WEHI-274.3 cells. Supernatants of cultures of WEHI-274.3 cells contained an activity that stimulated 3T3 fibroblasts to release an activity that stimulated the growth of the WEHI-274.3 cells. The 3T3-stimulatory activity released by the WEHI-274.3 cells was neutralized completely with an antiserum specific for murine IL-1 alpha, but not with antiserum specific for IL-1 beta. Moreover, WEHI-274.3 cells both in vitro and in vivo contained high levels of IL-1 alpha and IL-1 beta mRNAs. The leukemia-stimulatory activity released by the 3T3 cells was neutralized by an antiserum specific for GM-CSF. We postulate that the IL-1 alpha constitutively released by the WEHI-274.3 cells stimulates the production of GM-CSF from host cells such as fibroblasts or endothelial cells. A similar paracrine mechanism of growth stimulation may occur in acute myeloid leukemias in humans.     

Acute myelogenous leukemia of the Wistar/Furth rat: establishment of a continuous tissue culture line producing lysozyme in vitro and in vivo.

A transplantable myelogenous leukemia of an inbred Wistar/Furth rat has been established in tissue culture and cloned. The resulting transplantable leukemia line demonstrates in vitro doubling time of 20 hr, colony-forming efficiency of 5% in liquid and methylcellulos-containing medium, and a saturation density of 3.0 x 106 cells/sq cm in liquid medium. Following intraperitoneal inoculation, newborn rats developed solid tumors, ascities, and leukemia with ld50 of5 x 103 cells and mean latency of 60 days. The tumor cell morphology was consistent with that of acute myelogenous leukemia. Histochemical staining for myeloid enzymes revealed no evidence of myeloperoxidase, esterase, or leukocyte alkaline phosphatase; however, fluorescent antibody staining for lysozyme was markedly positive. Serum, urine, and ascitic fluid from rats with transplanted leukemia also contained elevated levels of lysozyme. There was no detectable type-CRNA virus production by this cell line after as long as 100 days in vitro. This inbred rat myelogenous leukemia should provide a useful model for studies of chemotherapy and immunoltherapy of human acute myelogenous leukemia.     

Induction of differentiation of myelomonocytic leukemia cells by mitoxantrone

We studied the effect of mitoxantrone (MIT) on the proliferation and differentiation of murine myelomonocytic leukemia cell line WEHI-3B (D+), human myelocytic leukemia cell line HL-60 and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). In a liquid culture, growth inhibition and differentiation of WEHI-3B (D+) cells to mature myeloid cells and a reinforcement tendency of the induction of nitroblue tetrazolium reducing capacity as well as of ASD chroloacetate esterase staining was observed by the treatment with MIT. Fresh ANLL cells classified as M4 were induced by MIT to undergo terminal differentiation to macrophage-like cells. Since at concentrations of WEHI-3B (D+) cells of less than 1 x 10(5)/ml induction of differentiation was observed due to MIT, it is suggested that its mechanism of action differs from that of the induction of differentiation by granulocyte colony-stimulating factor and is due to the direct action of MIT.     

Serological and biochemical characterization of surface antigen expressed on human myeloid cells and identification of six distinct antigen systems

Six distinct surface antigens selectively expressed on human myeloid cells were defined with ten murine monoclonal antibodies. Four antigens (GAp95, GAp170, GAp160-220, GAp145-200) are expressed on protein molecules and one antigen (GANGL) is detected in neutral glycolipids. The biochemical nature of the remaining one (GA8.9) is unknown. GAp95 and GANGL are expressed on relatively mature myeloid cells, whereas GAp160-220 is expressed on immature myeloid cells. GAp170 and GAp145-200 are expressed on a wide range of myeloid cells. GA8.9 was found on NKM-1, a cell line of acute myelomonocytic leukemia, and also on some acute nonlymphocytic leukemia cells.     

Disseminated extramedullary myeloid tumor of the gallbladder without involvement of the bone marrow

Extramedullary myeloid tumors (myeloid sarcomas) are rare neoplasms that are composed of myeloid precursors. They usually arise concurrently with a diagnosis of acute myeloid leukemia, chronic myeloid leukemia, or other myeloproliferative disorders. They may also indicate relapsing disease in a patient with a prior history of leukemia or myeloproliferative disorder. We present our findings of a 63-year-old female diagnosed with extramedullary myeloid tumor first presenting in the gallbladder. She subsequently developed respiratory failure; pre- and postmortem bone marrow studies were negative for leukemia by morphology, flow cytometry, and karyotypic analysis. However, the myeloid neoplasm was disseminated throughout most of her remaining organs. Immunohistochemical stains of the cells indicated a neoplasm of myelomonocytic derivation (CD4, CD43, CD45, CD68, myeloperoxidase, and lysozyme positive). To our knowledge, this is the first report of an extramedullary myeloid neoplasm of the gallbladder with disseminated disease without involvement of the bone marrow.     

Suppression of lymphocyte activation and functions by a leukemia cell-derived inhibitor.

An inhibitor isolated from the serum-free culture medium of human myeloid leukemic HL-60 cells was able to suppress mitogen- and alloantigen-stimulated proliferative responses of normal lymphocytes in a dose-dependent manner. In vitro production, concentration, and purification by column chromatography and electrophoresis revealed that the inhibitor was produced constitutively, required RNA synthesis, and had a molecular weight in the range of 40,000-60,000. The inhibitor was also produced in vitro by myeloid leukemia cells isolated from patients with acute myelogenous leukemia. In a similar manner, the inhibitory material suppressed proliferative responses of allogeneic and autologous lymphocytes. Suppression was accompanied by drastically reduced production of interleukin 2 and lymphokines, which regulate differentiation of myeloid leukemia cells, and suppression was reversed by addition of exogenous interleukin 2. The inhibitor did not suppress clonogenic proliferation of normal granulocytes and macrophages suggesting that inhibition of production or interference with interleukin 2 activity as a possible mechanism. These interactions between leukemia cells and lymphocytes have shed new light on the immunosuppression and growth advantage of leukemia cells. Inhibitory activity of HL-60 cells was diminished after they were induced to differentiate, indicating that differentiation induced by lymphokines may be an effective means of controlling leukemia.     

OMA-AML-1: a leukemic myeloid cell line with CD34+ progenitor and CD15+ spontaneously differentiating cell compartments.

OMA-AML-1 was established from a patient with acute myelomonocytic (M4) leukemia at fifth relapse when blasts were greater than 85% CD34+, CD15-. Leukemic cells were established in suspension culture and independently grown as subcutaneous tumors in SCID mice. Cells growing in suspension culture underwent differentiation by phenotypic and morphologic criteria. In contrast, cells grown as subcutaneous solid tumors in SCID mice maintained progenitor cell characteristics with high-density CD34 expression and lack of morphologic differentiation. A tendency toward differentiation to CD15+, CD34- cells in vitro and self-renewal of CD34+, CD15- cells in vivo was consistently demonstrated regardless of whether cells were initially grown in vitro or in vivo. The cell line maintains both a CD34+, CD15- progentitor cell pool and a non-overlapping, CD15+, CD34- differentiating cell compartment after more than 1 year in continuous culture. Cell cycle analysis and cloning experiments were consistent with terminal differentiation occurring in the CD15+, CD34- population. The cell line shows concentration-dependent proliferative responses to interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6, but not to granulocyte CSF (G-CSF). OMA-AML-1 appears to mimic several features of normal myeloid hematopoiesis and should prove useful for the study of normal and malignant myeloid differentiation.     

Induction of cytotoxic T lymphocytes by dendritic cells pulsed with murine leukemic cell RNA

Peptide-pulsed dendritic cells can stimulate T cells showing specific cytotoxicity in chronic myelogenous leukemia. We tried to induce a specific cytotoxic T-cell response stimulated by RNA-pulsed dendritic cells in acute myelogenous leukemia. The total RNA of WEHI-3BD+, a myelomonocytic leukemia cell line derived from BALB/c mice, was transfected into dendritic cells induced from bone marrow nucleated cells of BALB/c mice with granulocyte macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) using liposome. RNA-pulsed dendritic cells were injected into the peritoneal cavity of BALB/c mice, and splenic T cells were isolated for antigen-stimulated proliferation and leukemia-specific cytotoxicity assay. Cultured bone marrow nucleated cells expressed dendritic cell markers including MHC class II antigen, CD80, CD86, and CD11c. T cells stimulated by RNA-pulsed dendritic cells showed enhanced proliferation than those stimulated by unpulsed dendritic cells (P = 0.05) and showed dose-dependent specific cytotoxicity against WEHI-3BD+ cells. We concluded total RNA-pulsed dendritic cells could induce a specific T-cell cytotoxicity in acute myelogenous leukemia.     

Diisopropylfluorophosphate binding proteins (serine hydrolases) from normal and leukemic hematopoietic cells

Normal and leukemic hematopoietic cell lysates were labeled with [3H]-diisopropylfluorosorophosphate ([3H]-DFP), an active site inhibitor of serine hydrolases. The labeled proteins in the lysates were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by counting of gel segments for radioactivity. The results indicate the presence of distinct [3H]-DFP binding patterns for different normal and leukemic hematopoietic cells; significantly lower labeling in normal or leukemic lymphoid cells compared to myeloid or monocytoid cells; lower labeling in acute myeloblastic leukemia (FAB-M1) as compared to acute myelomonocytic leukemia (FAB-M4), chronic myelomonocytic leukemia or monocytes and an increase in [3H]-DFP binding with cell maturation along granulocytic series. Thus, these patterns could be useful in discriminating acute lymphoblastic leukemia from myeloid/monocytoid types of leukemia and for following maturation of myeloid cells, and perhaps for studying functional or maturation defects in hematopoietic cells in other pathological conditions.     

Clonal trisomy 8 is associated with myeloid phenotype rather than the neoplastic transformation in acute leukemia

Our studies of an acute leukemia with a clonal t(1;11) marker have demonstrated a conversion from pre-B cell to myelomonocytic phenotype associated with the acquisition of trisomy 8. This finding suggests that trisomy 8, a frequent clonal abnormality in acute myeloid leukemias, may be associated with the myeloid phenotype rather than the neoplastic transformation. A permanent myelomonocytic cell line, designated UF-SK1, has been established from leukemic cells with a 47,XX,+8,t(1;11) (p31;q25) karyotype and shown to have myelomonocytic characteristics, including phorbol ester-induced differentiation to macrophages. This new cell line will be a valuable tool in the study of leukemogenesis and lineage commitment.     

Increased amplification of abl oncogene in K562 cells after passage in nude mice

Human chronic myelogenous leukemic cells K562, carrying the Ph+ chromosome and an amplified abl oncogene, were injected subcutaneously in nude mice and gave rise to myeloid solid tumors after 4-5 weeks. DNA from the cells of the solid tumors was analyzed by Southern blotting using v-abl and actin probes. Increased amplification of human abl oncogene, but not of the actin gene was found. This suggests a clonal selection of a cell population with more copies of the abl oncogene. This in vivo selection appears to increase the tumorigenicity of K562 cells since, in a second transplantation in nude mice, we observed a shorter period of latency before tumor appearance (3 weeks) when compared to the initial transplantation.     

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.     

Growth of human myeloid leukemias in the human marrow environment of SCID-hu mice

It has been shown previously that multilineage human hematopoiesis is maintained within human fetal bone marrow (BM) fragments implanted into severe combined immunodeficient (SCID) mice. We describe here an application of this animal model, the SCID-hu mouse, to the study of human myeloid leukemias. BM cells from 8 patients with various types of myeloid leukemias were injected directly into human bone grafts in the SCID-hu mouse. Cells from 7 patients grew in the human marrow without spreading to the mouse marrow. Cells from 6 of these patients were successfully transferred in vivo to secondary SCID-hu recipients. The surface phenotype and the cytologic features of the leukemia cells were conserved during passage in vivo. Thus, human myeloid leukemia cells could be reproducibly propagated in the human marrow environment in SCID-hu mice. The differentiation of promyelocytic leukemia cells in the SCID-hu mice was induced by all-trans retinoic acid, suggesting that the biologic features of the leukemia cells were maintained as well. Finally, evidence for a leukemic progenitor cell population in one case of acute myelogenous leukemia was provided with this system. This model may provide a useful tool for studying the biology of human myeloid leukemia as well as for evaluating new therapeutic modalities for myeloid leukemias.