Bismarck B. Lozzio

PROPERTIES AND USEFULNESS OF THE ORIGINAL K-562 HUMAN MYELOGENOUS LEUKEMIA CELL LINE

THE K-562 CELL line was originally established in our laboratory in 1970 from a pleural effusion of a patient with CML in terminal blastic crisis and has been characterized as a highly undifferentiated cell of the granulocytic series [17, 23-25, 28]. In view of the fact that this laboratory was the original source of K-562 cells and the progenitor of all sublines now in existence and doubts have recently been cast [1, 2] on the nature of the K-562 cell line, an update of the structural and functional features of the original cell line appears to be warranted. This cell line has been maintained in our tissue culture unit for nearly 9 y as continuous serial culture passages and as a series of stock passages preserved frozen in liquid nitrogen. Data on the K-562 cells not reported previously are presented. The applications and usefulness of this unique and reliable source of myelogenous leukemia cells of human origin in approaches to the immunotherapy of leukemia have also been included in this review. The information provided herein should serve as the standard reference information to compare with the characteristics of sublines derived from the original K-562 cell line and currently maintained in literally hundreds of laboratories around the world. To fully assess the information of the original K-562 cell line, the results of studies done by others on sublines of K-562 have purposely been excluded.

A Multipotential Leukemia Cell Line (K-562) of Human Origin

Abstract The K-562 leukemia cell line, originally established in our laboratory, has been characterized as an early precursor of the granulocytic series with a block for differentiation. Since K-562 blasts did not differentiate when cultured for 7-8 days in liquid media or 14-16 days in agar-gel an attempt was made to stimulate their potential for spontaneous differentiation by prolonging the time in culture. Inducers of differentiation were not added to the cultures and the cells were studied when they reached the steady state rather than during exponential growth. The cultivation of K-562 cells for 10 to 11 days in media gradually depleted of the essential nutrients needed for cell division induced their differentiation into early precursors of the monocytic, granulocytic, and erythrocytic series. Thus, the peroxidase reaction for hemoglobin demonstrates benzidine-positive material limited to the region of the Golgi apparatus. Analysis of the hemoglobin by isoelectric focusing indicated major bands in the region of embryonic hemoglobin. Most cells (80-90%) give a strong reaction for α-naphthyl acetate esterase typical of monocytes and as many as 30 to 40% of the cells have abundant red cytoplasmic granules of naphthol AS-D chloroacetate esterase characteristic of granulocytic precursors. Myeloperoxidase activity was found in 5 to 10% of the cells. Polyploid cells (5-8%) and early myelomonocytic precursors have PAS-positive material, were stained with Sudan black, and possessed abundant acid phosphatase. The data support the conclusion that K-562 is, indeed, a multipotential leukemia cell line of human origin.

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.

Regulators of cell division: endogenous mitotic inhibitors of mammalian cells.

Publisher Summary This chapter deals with the antimitotic substances found in a variety of tissues and sera of humans and animals, as well as with inhibitors produced by cultured cells. It appears that cultured cells release substances which in turn inhibit cell division when the appropriate concentration in the medium is reached. Thus specific and nonspecific inhibitors of cultured cell growth have been reported. Some normal cells appear to produce an inhibitor of the proliferation of oncogenic virus-containing cells which are unable to synthesize a similar antimitotic substance. The growth of normal fibroblasts may be controlled by contact inhibition, protein factors present in the serum added to the medium, and attachment to rigid surfaces (anchorage-depending growth). Extracts from mice and chick embryos have been found to suppress cell growth in vivo andin vitro, respectively. The administration of mouse embryonic and placental extracts inhibited the growth of 70% of spontaneous and transplanted tumors. Since the treatment was mainly effective on carcinomas and not on sarcomas, the extract appeared to have some tissue specificity. Low-molecular weight inhibitors of normal cells have also been obtained from chick embryos. Specific and nonspecific mitotic inhibitors have been partially purified from mammalian and amphibian kidneys. The existence of a chalone, probably protein in nature, has been reported in amphibian kidneys. It may control growth and differentiation from the early stages of the pronephros, as well as kidney cell renewal in adult life. Numerous growth inhibitors isolated from the liver were active against various tumor cells both in vitro and in vivo.

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 γ‐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, γ‐globulins, a mixture of the two, or conjugates of drugs with normal goat γ‐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 γ‐globulins without the loss of drug or antibody biological activity. Although the simultaneous administration of chlorambucil and γ‐globulins and conjugated drug γ‐globulins reduced the growth of myelosarcomas considerably, the immune γ‐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.

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.

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 107 malignant cells. K-562 cells (105) 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.

Immunotherapy of metastases of human neoplastic cells grown in immunodeficient mice

SummaryHereditarily athymic (nude) and asplenic-athymic (lasat) mice were inoculated neonatally with 107 K-562 pluripotential leukemia cells of human origin. Meningeal infiltration and/or multiple metastases were found in the lungs, kidneys, and lymph nodes in nearly 60% of mice. Twice as many lasat mice as nude mice had meningeal and lymph node infiltrations. This result indicates that the spleen of nude mice influences the infiltrations and/or the distribution of metastases. Metastases of K-562 cells were found as early as 10 days and as late as 115 days of age. Goat immune gamma (γ)-globulin, prepared from antiserum to K-562 cells and absorbed with peripheral leukocytes and bone marrow cells from normal individuals, markedly diminished the incidence of metastases of K-562 cells. About 16% of the mice treated with immune γ-globulin had metastases in the lungs only. All mice receiving the immune γ-globulin had peripheral monocytosis and lymphocytosis as well as a hyperplasia of the bone marrow monocytic series. Immune γ-globulin may lyse heterotransplanted leukemia cells by direct binding to leukemia cells in the presence of complement and/or may activate antibody-dependent effector cells, for example macrophages or killer cells, which would destroy the transplanted leukemia cells.

Alteration of bone marrow-thymus cell synergism in hereditary asplenic and adult splenectomized mice.

Summary The cooperation between bone marrow (B) and thymus (T) cells was markedly impaired in mice with congenital as-plenia. The deficiency of IgM producers could not be corrected by neonatal transplantation of spleen cells. The use of T and B cells from splenectomized donors results in a marked shift from 19S to 7S antibody forming cells.

Induction of Interferon in Hereditarily Asplenic Mice With and Without a Neonatal Spleen Cell Transplant

Summary Interferon production in hereditarily asplenic and normal mice following intravenous injection of Newcastle disease virus was compared. Serum from asplenic mice showed a significantly lower interferon level than normal littermates. A neonatal spleen cell transplant markedly enhanced interferon production in asplenic mice to the extent that they were able to produce amounts of interferon approximately the same as normal littermates with spleen.