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.

Hereditary asplenic-athymic mice: transplantation of human myelogenous leukemic cells.

A new animal model characterized by hereditary athymia and asplenia was used as a recipient of chronic myelogenous leukemic (CML) cells with the Philadelphia (Ph1+) chromosome. Transplanted CML cells form solid vascularized tumors containing cells similar to those seen in the patient in a long-term culture. Cells taken from the tumors were nearly triploid, retained all human chromosome markers, and had the same antigenic determinants(s) as cells in culture.

Hereditary renal disease in a mutant strain of rats.

Disease of the kidney developed in breeding stock of Gunn rats. The renal lesion is the result of a new mutation. The genetic defect is inherited as an autosomal dominant trait and is apparently lethal in the homozygous condition. The abnormality manifests itself as a congenital hydronephrosis with related cystic changes in the kidney.

Human myelogenous (Ph+) leukemia cell line: transplantation into athymic mice.

Chronic myelogenous leukemia (CML) cells from line K-562, containing the Philadelphia (Ph1) chromosome, were transplanted into nude mice and grew as solid vascularized tumors containing cells like those seen in the patient and in the cultures. Cells taken from the tumors were near triphoid and retained all human chromosome markers in culture.

Reproducible metastatic growth of K-562 human myelogenous leukemia cells in nude mice.

A reproducible metastatic growth of K-562 human myelogenous leukemia cells occurred in immunodeficient athymic (nude) mice. Although previous studies have shown that K-562 cells grow as local subcutaneous myelosarcomas which continuously release leukemia cells into the systemic circulation in adult mice, metastases were not observed. However, the subcutaneous "priming" of newborn nude mice resulted in the metastatic proliferation of leukemia cells in the lungs, kidneys, brain, and lymph nodes. Three sc injections of 5 X 10(6) K-562 cells on days 1, 7, and 14 of life produced metastases in 51% of the mice. When the initial series of injections was followed by iv injections on days 35 and 42, the incidence of metastases increased to 67%. Karyotypes demonstrated that the tumor cells retained the same human chromosome markers as those in the human patient and tissue culture. These procedures may provide a model for study of the mechanisms of metastases and for chemotherapeutic and immunotherapeutic trials against metastases of neoplasms of human origin.

Characterization of an antigen from the myelogenous leukemia cell line K-562.

A protein was solubilized from the myelogenous leukemia cell line K-562 WITh 3 M KCl that specifically inhibited the antibody-dependent, complement-mediated cytolysis of 51Cr-labeled K-562 cells by a monkey antiserum to K-562. When the crude 3 M KCl extract uas fractionated with ammonium sulfate, an eightfold increase in specific activity (U inhibition/mg protein) resulted. This purified fraction migrated as a single protein band after polyacrylamide gel electrophoresis (PGE) with no detectable carbohydrate or lipid. The molecular weight of the denatured protein determined by sodium dodecyl sulfate-PGE was 77,000, similar to that of the native protein (80,000) determined by Sephadex exclusion chromatography. The protein was stable at pH 6-8, with an apparent isoelectric point between pH 5 and 6. In addition to being irreversibly denatured at pH 5 or less, it was unstable at osmolarities below 0.25 M (NaCl). It was denatured at temperatures of 56 degrees C or above. Normal human peripheral blood leukocytes were extracted similarly with 3 M KCl and fractionated with ammonium sulfate. Neither the crude preparation nor any fraction purified as described for the specific antigen inhibited the cytolytic assay, which indicated at least a quantitative lack of the protein on the surfaces of normal leukocytes.

Cytotoxicity of antisera to a myelogenous leukemia cell line with the Philadelphia chromosome.

Rabbit antisera to myelogenous leukemia (ML) cells were raised; ML cells from line K-562 that has the Philadelphia (Ph) chromosome were used as antigen. Antibodydependent, complement-mediated cytotoxicity was demonstrated by the trypan blue test and Cr release assay for cultured ML cells, whereas no cytotoxicity was demonstrated for cells from B (SB) and T (MOLT 4) lymphoblastoid cell lines. The antisera showed no cross-reactivity for normal human peripheral leukocytes or purified granulocytes. A low level (less than 8%) of cytotoxicity was directed against cell membrane associated fetal bovine serum proteins. Absorption of the immune serum with normal human bone marrow cells of first trimester human whole embryo cells reduced the cytotoxic titer to a similar extent; this suggested the possibility of crossreactivity between ML cells and fetal antigen(s). However, the ML antigen(s) was unrelated to carcinoembryonic antigen (CEA), since absorption with CEA had no effect on the serum cytotoxic titer. The anti-ML sera were cytotoxic for cells taken from 10 patients with chronic myelogenous leukemia and from 3 with acute myelogenous leukemia. In contrast, the leukocytes of 1 of 4 patients with acute lymphocytic leukemia, and 3 of 7 with chronic lymphocytic leukemia shared similar antigenic determinants as demonstrated by cytotoxicity tests. The significance of the cross-reactivity of some lymphatic and ML cells may be the result of the use of rabbit sera that did not distinguish antigens common to both granulocytic and lymphocytic cells, or it may reflect an "immature" or "blastic" antigen present on many leukemia cells.

High incidence of mammary tumors in mice with inherited asplenia carriers for the nude gene.

A colony of mice suffering from dominant hemimelia associated with agenesis of the spleen has been developed and characterized during the past 7 years. The hereditarily asplenic (Dh/+) mice have a very low incidence (9%) of spontaneous mammary tumors (SMT). Asplenic (Dh/+) females were mated with mice homozygous (nu/nu) for hereditary athymia (nude) having a BALB/c background. BALB/c females heterozygous for the nu gene and with spleen (nu/+,+/+) have a moderate incidence (12%) of SMT, whereas nu/+,Dh/+ breeders have a drastic increase in the incidence of SMT to 46% when bred under identical conditions. Since all parent strains have a very low incidence of SMT, it appears that the spleen agenesis is a major factor accounting for an earlier and higher incidence of SMT in hereditarily asplenic (nu/+,Dh/+) mice than in normal (nu/+,+/+) siblings. The SMT express mammary tumor virus antigen(s) and possess estrogen, progesterone, and glucocorticoid receptors. The SMT rapidly metastasize and kill the host within 30 to 45 days. The BALB/c asplenic mice with SMT represent a unique model relevant to human breast cancer and for study of the function of the spleen in the development of solid tumors in general and of SMT in particular.

Absence of thymus-derived lymphocyte markers in myelogenous leukemia (Ph1+) cell line K-562.

The myelogenous leukemia cell line K-562 with a Ph1+chromosome, derived from a patient with chronic myelogenous leukemia in terminal blastic crisis, is not a bone marrow-derived lymphoblastic cell line, because the cells neither produce immunoglobulins nor possess complement receptors. Since it has been suspected that blasts found in some patients with chronic myelogenous leukemia in blastic crisis might be thymus-derived cells, we have studied several parameters to demonstrate that K-562 cells are not thymus-derived lymphoblasts. The results of this study show: (a) no cross-reactivity of antisera to K-562 cells with normal human thymocytes; (b) lack of cytotoxicity of a specific horse anti-human thymocyte globulin for K-562 cells; (c) failure of the treatment of K-562 cells with bovine thymosin to induce antigenic determinant and erythrocyte rosette receptors on K-562 cells; (d) presence of receptors for the Fc portion of immunoglobulin G; (e) absence of terminal deoxynucleotidyl transferase; and (f) cytotoxicity of monkey antiserum to K-562 cells for malignant thymus-derived cells (Molt-4). However, absorption with Molt-4 cells abolished the cross-reactivity with Molt-4 cells, whereas 60% of the antibody to K-562 cells remained in the immune serum. Studies of DNA polymerase activities revealed that K-562 cells have levels of polymerase alpha and beta, like other proliferating cells, and an RNA-dependent DNA polymerase activity, presumably representing polymerase gamma.

Hematopoiesis of hereditarily asplenic-athymic (lasat) mice.

The hematopoiesis of athymic-asplenic (lasat) mice was compared with that of normal, asplenic, and athymic littermates with the same strain background. Erythrocyte blood volume, number and survival time were normal when related to the body weight of the animals. Peripheral blood showed leukopenia with absolute and relative lymphopenia, resembling the athymic rather than the asplenic pattern. The bone marrow was hypocellular as a consequence of a decrease in both lymphocytes and erythroid precursors, while thrombocytopoiesis and granulcytopoiesis-monocytopoiesis were essentially normal. Although the percentile value of femoral stem cells was high, their absolute number was, in fact, reduced by 35% as a result of the bone marrow hypocellularity. When lasat bone marrow cells were injected into normal, lethally irradiated mice, a rapid erythropoietic recovery was observed, whereas the restoration of the granlocytic compartment was impaired. It was concluded that: 1) lasat mice depict a normal hematopoiesis in spite of the congenital absence of the thymus and the spleen; 2) bone marrow stem cells may be defective when administered to lethally irradiated hosts; and 3) the athymic status predominates over the asplenic one.

Infleuence of a neonatal spleen transplant on hematopoiesis of hereditarily asplenic mice.

Hereditarily asplenic mice and normal litternates were transplanted subcatneously with 20x10(6) congenic spleen cells at birth. Eighty per cent of the recipients developed subcutaneous nodules closely resembling the spleen structure seen ia a normal mouse. The growth of the graft was associated with hyperplasis of the majority of the bone marrow cellseries and an increased number of stem cells (CFU-spleen) and agar colony forming cells (CFC) per femoral shaft. Particularly noticeable was the hyperplasia of the granulocytic series which paralleled the increase of CFC number and of serum colony stimulating factor. The presence of the graft did not modify the erthrocytic seroes and the leukocytics of asplenic mice but reduced the average platelet count. It is xonxluded that the growth of the transplant was associated with the hematopoietic alteration described. These results would also indicate humoral or cellular influences of the spleen on the release of or production of marrow cells.

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.

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.

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.