Interaction of the heterozygous nude gene with the asplenia trait in mammary tumorigenesis.

The BALB/c mouse strain has been shown to contain endogenous mouse mammary tumor virus (MMTV) proviral sequences. However, no exogenous MMTV particles have been detected in their tissues. Female BALB/c mice from our colonies exhibit a very low incidence of spontaneous mammary tumors (SMT); less than 1% at up to 20 mo of age. Immunodeficient BALB/c mice heterozygous for the nude gene (nu/+, +/+), for the dominant hemimelia gene associated with asplenia (+/+, Dh/+), or for both traits (nu/+, Dh/+) have been examined for SMT incidence and the presence of MMTV proviruses. Based on restriction digestion with Eco RI, Bam HI, and Pst I, the immunodeficient mice have an MMTV provirus copy number and organization identical to the BALB/cCrgl strain. This MMTV DNA pattern is distinct from the MMTV proviruses in C3H/He, C57BL/6J and CBA/CaJ mice, which were parental strains of the immunodeficient mutants. Normal female BALB/c or BALB/c heterozygous for the asplenic trait do not develop significant numbers of SMT at up to 19 mo of age. In contrast, an incidence of 23.8% and 57.7% SMT was observed in BALB/c nu/+ heterozygotes, and in BALB/c nu/+, Dh/+ heterozygotes, respectively. These results indicate that agenesis of the spleen, concomitant with the presence of the heterozygous nude gene, contribute to a high incidence of SMT in the low-SMT BALB/c mouse strain.

Proliferation and differentiation of human myeloid leukemic cells in immunodeficient mice: electron microscopy and cytochemistry.

To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross-reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.

K562 human leukemia cell passages differ in embryonic globin gene expression.

K562 is a human leukemia cell line inducible by a variety of agents for the synthesis of embryonic and fetal hemoglobins. We compared early and late passages to determine whether a change has occurred in globin synthetic pattern. Clone LA4, derived from passage 199 which had been frozen by Lozzio in 1973, was compared with clone RA6, derived from a line received from Rutherford in 1979. Globin synthetic pattern was determined by incubation with [3]leucine, separation of globins by Triton-X100 polyacrylamide gel electrophoresis, and analysis by fluorography. For RA6, hemin-induced synthesis was greatest for zeta globin but minimal for epsilon globin, whereas for LA4 it was greatest for epsilon globin but minimal for zeta globin. Both lines are pseudotriploid with three No. 11 and three No. 16 chromosomes. However only RA6 has a translocation involving the short arm of chromosome 11 which contains the locus of the beta globin gene cluster. However, translocation-associated deletion does not simply explain the deficient inducibility of epsilon synthesis because G gamma and A gamma globins, whose genes are linked to the epsilon gene, are similarly inducible in the two lines.

Proliferation of human malignant hematopoietic cells in immunodeficient mice: suppression by antibody to pluripotent K-562 leukemia cells involves direct cytolysis and effector cells.

Six human hematopoetic cell lines were successfully heterotransplanted into athymic (nude) and asplenic-athymic (lasat) neonatal mice. The tumors arising from leukemia and lymphoma cells could then be serially transplanted into adult nude mice. Seven days after the fourth serial mouse passage, each mouse was treated with goat immune gamma globulin against K-562 cells. One control group was treated similarly, but with nonimmune (normal) gamma globulin, while another control group was not treated. The goat gamma globulin was not toxic for nude and lasat mice, and the immune, but not the normal, gamma globulin suppressed local subcutaneous growth of myelosarcomas, lymphosarcomas, and Burkitt lymphoma cells. On the other hand, the growth of lung, breast, and prostatic carcinomas and a melanoma of human origin were not altered by the immune gamma globulin. Since suppression of cell growth occurred equally well in decomplemented mice, a complement-mediated cytotoxicity apparently cannot be considered as responsible for the abrogation. The Fab fragment of the immunoglobulin did not suppress the growth of the myelosarcomas. We conclude that antibody suppression of the in vivo proliferation was specific for malignant hematopoietic cells and that the Fc portion of IgG is necessary for in vivo cytolysis of leukemia cells. The most probable mechanisms are direct antibody cytolysis and antibody-dependent macrophage-mediated cytotoxicity.

Immunotherapy of human leukemia with antibody to pluripotential K-562 stem cells.

Gamma (gamma) globulin was fractionated from the serum of a goat immunized with the pluripotential leukemia cell line K-562. The lyophilized gamma-globulin preparation, termed leukoglobulin, contained about 50% immune IgG and suppressed the proliferation of heterotransplanted leukemia and lymphoma cells of human origin. The main aims of this study were to evaluate the potential therapeutic value of leukoglobulin and to determine its toxicity in humans with terminal leukemia and patients whose disease was unresponsive to current therapy. Two patients with CML, one with AMML, four with ALL, and one with AML were treated once a week for up to 5 weeks with leukoglobulin intravenously at doses ranging from 2 to 29 mg/kg. Leukoglobulin was well tolerated with minimal adverse effects and produced an initial mobilization of blasts from the bone marrow, spleen, and other organs with a parallel increase in the number of blasts in the systemic circulation. Subsequent injections of leukoglobulin led to a sharp decrease and the eventual eradication of blasts from the peripheral blood and bone marrow. Except in patients with CML, immature cells other than blasts also markedly diminished. The results of the clinical trials indicated a synergism with or potentiation of most chemotherapeutic agents used. Two possible uses for a combination of leukoglobulin and antileukemic drugs are indicated by the results we report here; drug-antibody synergism for cases showing no response to chemotherapy alone or leukoglobulin given immediately after chemotherapy is administered to eliminate residual leukemia cells. Alternatively, leukoglobulin can be given as a single therapeutic agent during the induction or maintenance phases of treatment to patients with leukemia resistant to other therapeutic combinations.

Relationship between the Fc receptor for IgG and the specific associated antigen of the pluripotential leukaemia cell line, K-562.

The cells in the K-562 line have been shown to express Fc receptors as demonstrated by rosette formation with sheep erythrocytes (E) sensitized with haemagglutinin (EA). Rosette formation is inhibited by prior incubation of the cells with goat or monkey anti-K-562 serum, gammaglobulin fraction, or Fab fragments. Alkaline aggregated human IgG also inhibits rosette formation. Furthermore, formed rosettes can be dissociated by goat anti-K-562 gammaglobulins indicating that the binding of the ligand to one entity interferes with the binding to the other. We also found that treatment of K-562 cells with specific anti-K-562 globulin leads to patching and capping of surface antigen. After patching and capping, the cells will not form rosettes. These findings suggest that the Fc receptor and the K-562 associated antigen are either located very close to one another or are the same entity.

Absence of morphological, chromosomal and antigenic changes in the K-562 cell line growing as localized or disseminated tumours in nude mice.

Transplantation of K-562 cells into adult and newborn nude mice led to the development of localized s.c. and disseminated myelosarcomas, respectively. This age-associated, changing pattern of in vivo proliferation of K-562 cells derived from a single aliquot was consistently repeated throughout sequential passages. The only variable in this experimental system was the age of the recipient mice. Not only did the mice have an identical genetic background, but also the transplanted K-562 cells were derived from a single culture passage. As shown by cytological and histological examinations, the characteristic morphology and percentage composition of the subpopulations of the K-562 cell line were preserved in successive in vitro and in vivo passages. The K-562 cells had no prevailing phenotypic traits which could be associated with the growth either in the s.c. tissue or in the viscera. Furthermore, the cells maintained the human karyotype, including their typical chromosomal abnormalities and antigenic determinants, as demonstrated by the binding of a specific antibody, throughout all passages. Our results demonstrate that heterotransplanted K-562 cells may change their behaviour in vivo without undergoing modifications associated with different types of growth. These findings would indicate that the ability of neoplastic cells to proliferate in various environments (metastases) is not the consequence of predetermined cellular characteristics but is functionally conditioned.

Antibody lysis of human hematopoietic cells in the absence of complement and effector cells.

The incubation of K-562 leukemia cells with specific goat immunoglobulin resulted in gradual cytolysis in the absence of complement and effector cells. Optimal lysis (100%), reached in 3-4 days, occurred when the concentration of the antibody in the culture medium was about 20 micrograms/ml containing an initial inoculum of 15,000 cells. A lower concentration (10 micrograms/15,000 cells/ml) led to partial cytolysis; the surviving cells, cultured in fresh medium, were still vulnerable to antibody lysis (30 micrograms/ml), thus indicating the lack of an apparent resistant cell population. The amount of free antibody in the culture medium diminished rapidly and very little or none was detectable after 2-3 hours of incubation with K-562 cells. The immune gamma-globulin also inhibited colony formation of K-562 cells in soft agar. The immune gamma-globulin, absorbed extensively with normal blood cells, to pluripotent K-562 cells, cross-reacted with several human malignant hematopoietic cell lines tested. These cells included T-lymphoblasts (JM and MOLT-4), promyelocytes (HL-60), Burkitt's lymphoma cells (RAJI), myeloblasts (KG-1 and KG-1a), and multiple myeloma cells (K-737). The absorption of the immune gamma-globulin with an equal number of cells from each of the cell lines assayed diminished but did not completely remove the antibody responsible for cytolysis of K-562 target cells. Thus, the K-562 leukemia blasts appear to possess common and specific antigens that may be only partially expressed on other committed leukemia cell lines studied. For this reason, the cytolytic activity of the immune gamma-globulin could only be removed by absorption with K-562 cells possessing a complete array of antigens solely present on pluripotential hematopoietic cells.

Identification and quantitation of embryonic and three types of fetal hemoglobin produced on induction of the human pluripotent leukemia cell line K-562 with hemin.

The hemoglobins synthesized by the pluripotent K-562 leukemia cell line of human origin after induction with hemin have been isolated by DEAE-cellulose chromatography and characterized by electrophoresis, high pressure liquid chromatography, and a radioimmunological assay. Six hemoglobin zones have been observed with the following likely compositions. Zone 1: alpha 2 epsilon 2, or HB Gower-2; zone 2: zeta 2 epsilon 2, or HB Gower-1; zone 3: zeta 2 gamma 2, or HB Portland-I; zone 4: Hb F, or alpha 2 gamma 2; zone 5: a mixture of acetylated HB Portland-I and Hb F; zone 6: Hb Bart's, or gamma 4. The embryonic Hbs (zones 1, 2, and 3) constituted 50%-75% of the total Hb present; the quantities varied from one experiment to the other. Both Hb Gower-1 and Hb Gower-2 were present. The gamma chain was heterogeneous and contained the G gamma, A gamma I, and A gamma T types in a ratio of about 4:2:1, indicating a heterozygosity for the Ile leads to Thr substitution at position gamma 75. The methodology used can be applied for additional studies evaluating quantitative changes in Hb types due to in vitro manipulations.

Arrest and extravasation of neoplastic cells. An electron microscopy study of serial sections at sequential stages.

The morphological aspects of the arrest and extravasation of malignant cells of human origin (K-562 cell line) in the lungs of athymic (nude) and asplenic-athymic (lasat) mice were studied by electron microscopy examination of serial sections. The specimens were obtained at sequential stages after the sc inoculation into newborn mice of 10(7) malignant cells. K-562 cells (10(5)) were also injected iv into control groups of nude and lasat mice to assess the influence of the route of inoculation on the in vivo behavior of K-562 cells. Our results demonstrated that K-562 cells were arrested and proliferated within the pulmonary capillaries without the participation of platelets or fibrin. The neoplastic cells extravasated by attrition and penetration of the endothelium (rather than by diapedesis) and continued to proliferate in the interstitial tissue of the lung, developing into neoplastic nodules. Following iv injection, K-562 cells induced the formation of platelet-tumor cell aggregates within the pulmonary capillaries. However, under these conditions, the neoplastic cells did not adhere to the endothelium nor did they proliferate or extravasate. These aggregates were flushed out by the circulation, restoring the permeability of the capillaries.

Relevance of lipids to heterotransplantation of human malignancies.

Although the transplantation of human neoplasms in immunodeficient mice is now a well-established procedure, the majority of primary malignancies cannot be successfully maintained for long periods of time in adult athymic (nude) and asplenic-athymic (lasat) mice. Various lipids such as cholesterol, cholesterol oleate, stearic and palmitic acid esters markedly depress the RES phagocytic activity and immunocompetence of mammals. In view of the immunosuppressive properties of certain lipids and in order to graft and grow as many tumors as possible, further studies into the effects of lipids on the growth of heterotransplanted human tumors is warranted. Lipids may enhance local growth and facilitate the development of metastases rarely seen in nude and lasat mice bearing xenogeneic cancer cells. Lipids may accelerate human malignant cell proliferation in mice by both depressing further the defense of host and modifying the cancer cell membrane. The relationship of lipids to the onset and progression of 'spontaneous' tumors in humans is not known.

Antibody-dependent cytolysis of the human leukemia cell line K-562 in the absence of effector cells or complement.

K-562 human leukemia cells grown in the presence of specific goat anti-K-562 gamma globulin, F(ab)'2, Fab', or Fab showed a decrease in DNA and protein syntheses and a loss of cell viability within several hours. Eventually all cells died and lysed within 2-5 days. These events occurred in the absence of added effector cells or complement. The effect on the cell was not due to proteolytic or other external lytic activity generated at the cell surface, since 51Cr-labeled bystander cells or antibody-sensitized bystander cells (chicken erythrocytes or K-562 cells) were not lysed when added to cultures of K-562 cells incubated with immune globulin. The anti-K-562 gamma globulin was not observed to patch or cap on K-562 cells, but the ability of bound immunoglobulin to fix complement decreased markedly (50% in 60 min), so that no complement-mediated cytolysis could be observed at 2 hr. However, the immunoglobulin, at least portions of it, reacted with fluorescein--labeled rabbit anti-goat gamma globulin for about 16 hr. 125I-labeled immune globulin appeared to be internalized and released into the medium as small degradation products. 125I-labeled Fab did not appear to be internalized, since it was released intact and more rapidly from cells than the intact globulin.

Induction of hemoglobin synthesis in original K 562 cell line.

Cells of the original human myelogenous leukemia cell line, K 562 were induced to synthesize hemoglobin by incubation with 0.05 mM hemin. Analysis by isoelectric focusing indicated major bands in the region of hemoglobin F, Bart's hemoglobin, and embryonics. Cytochemical analysis by the Lepehene peroxidase reaction for hemoglobin indicated an heterogeneous population of cells with respect to hemoglobin induction. Approximately 50% of the cells were positive for hemoglobin. The benzidine-positive material was present as granules in the region of the Golgi apparatus of large blasts with undifferentiated nuclei. There was no indication of normal maturation or of terminal differentiation into reticulocytes or erythrocytes.

Correlation if circulating immune complexes and disease status in patients with leukaemia.

The occurrence of soluble immune complexes (IC) was investigated in 177 serum samples from 92 patients with various leukaemias using the Raji cell immunoassay. In general, patients with myeloproliferative diseases had a higher incidence and higher quantities of IC than did patients with lymphoproliferative disorders. Elevated levels of IC were found in the sera of patients as follows: 17% with chronic lymphocytic leukaemia (mean value of 13.1 microgram/ml), 67% with acute lymphocytic leukaemia (54.1 microgram/ml), 65% with chronic myelocytic leukaemia (86.7 microgram/ml), 70% with acute myelocytic leukaemia (202.5 microgram/ml) and 56% with acute myelomonocytic leukaemia (41.9 microgram/ml). Patients in terminal blastic crisis of chronic myelocytic leukaemia had the highest levels, with a mean level of 1,364.1 microgram/ml. Serial samples were obtained, as available, from individual patients during the course of the disease in an attempt to relate severity with the incidence and quantity of IC. No significant correlation could be made between the occurrence or levels of IC and the presence of absence of systemic symptoms. Similarly, no correlations could be made between levels of IC and haematological parameters, infection, or therapy. However, the data does indicate a positive relationship between the levels of IC and the progressive state of the leukaemia, especially, the myelocytic leukaemias.

Evaluation of drug-antibody conjugates in the treatment of human myelosarcomas transplanted in nude mice.

Methotrexate, daunomycin, and chlorambucil were independently conjugated to immune goat gamma-globulins specifically raised to the Ph1 + chronic myelogenous leukemia cell line K-562. The drug-antibody conjugates were then tested against myelosarcomas made up of K-562 cells growing in nude mice and their efficacy was compared with that of the drug alone, gamma-globulins, a mixture of the two, or conjugates of drugs with normal goat gamma-globulin. Conjugation methods for methotrexate and daunomycin abrogate the antibody activity as indicated by the absence of complement-mediated cytotoxicity of the conjugates in vitro and the lack of effect on myelosarcomas in vivo. Simultaneous administration of either of these drugs and antibody partially abrogated the development of myelosarcomas. Chlorambucil-antibody conjugates, however, retained their cytotoxicity in vitro and were found effective in vivo. It is the first successful attempt to covalently bind chlorambucil to gamma-globulins without the loss of drug or antibody biological activity. Although the simultaneous administration of chlorambucil and gamma-globulins and conjugated drug gamma-globulins reduced the growth of myelosarcomas considerably, the immune gamma-globulins alone either reduced their weight to a larger degree or eliminated their growth completely. Results of this study indicate that myelosarcomas made up of K-562 cells grown in nude mice are good and reproducible models for testing various therapeutic agents. The advantage of using human cells proliferating in an in vivo environment brings experimental therapy one step closer to clinical trials.

Thymocyte reconstitution of athymic and athymic-asplenic mice: graft rejection and antibody synthesis.

Hereditarily athymic and athymic-asplenic mice were reconstituted with syngeneic thymocytes at various times prior to and after tumor challenge. These mice were able to reject heterotransplanted human tumors in as little as 19 days after thymocyte transfer but were unable to do so when the thymocytes were given 7 days after tumor implantation. The humoral immunity of athymic nude mice was fully restored as determined by the primary immune response to sheep erythrocytes (SRBC), a thymocyte-dependent antigen. Athymic-asplenic mice failed to respond to SRBC after reconstitution and this was attributed to the absence of the spleen in these mice.