Antibody-dependent cellular cytotoxicity (AAZZ) against leukemia cells--K-cell activity of human leukocytes

The activity of human leukocytes from the peripheral blood (PB) and bone-marrow (BM) to take the function as K-cells in the antibody dependent cellular cytotoxicity (ADCC) was tested in a 51Cr-test against mouse leukaemic cells and ALL cells covered with specific heterologous antibodies. Mononuclear PB-leukocytes and granulocytes of healthy donors and patients with leukemia and lymphoma in remission lysed murine and human leukaemic cells in the presence of specific antibodies. There was no lowering of K-cell activity of mononuclear PB-leukocytes of patients with leukaemia and lymphoma in remission under chemotherapy as compared with healthy donors and patients in remission without chemotherapy. There was a good correlation between the percentage of K-cell active mononuclear leukocytes in PB and BM. Attempts of fractionation with peripheral blood leukocytes of healthy donors resulted in the non-adherent mononuclear PB-leukocytes (lymphocytes) and granulocytes being effector cells in ADCC. To a high degree K-cell active lymphocytes could be identified in the non-B-fraction and only slightly in the fraction forming E rosettes.     

Cancer stem cells: lessons from leukaemia

Increasing evidence suggests that leukaemias are sustained by leukaemic stem cells. Leukaemia can indeed be viewed as aberrant haematopoietic processes initiated by rare leukaemic stem cells that have maintained or re-acquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of leukaemic stem cells and the molecular pathways underlying the transformation of normal cells into leukaemic stem cells. This report will review our current knowledge on leukaemic stem cells development and finally demonstrate how these discoveries provide a paradigm for identification of cancer stem cells from solid tumours.     

Mathematical model of acute myeloblastic leukaemia: an investigation of the relevant kinetic parameters

A simple model of acute myeloblastic leukaemia (AML) development is introduced, explicitly including cell growth, cell differentiation and cell-cell interaction. Each of these processes is described by a single model parameter. It is hypothesized that the leukaemic cell is characterized by an alteration of only one of these processes. The kinetic behaviour of the AML system is examined separately for possible alterations of each of the three parameters describing the three processes involved. It is shown that, on the basis of the existing data on AML kinetics, the alteration of the growth and cell-cell interaction parameters can be eliminated as a possible source of AML. Thus kinetic data support the modification of the differentiation process as the origin of the AML state. Further, the growth characteristics of normal and leukaemic cells in the presence of each other are analysed. It is shown how the initial growth of leukaemic cells depends on the difference in the differentiation of normal and leukaemic cells and how the same difference determines the decay of normal cells in the presence of the predominantly leukaemic population. Correlations between the kinetic parameters of the normal and leukaemic populations are suggested to characterize the leukaemic state.     

Mathematical Model of Acute Myeloblastic Leukaemia: an Investigation of the Relevant Kinetic Parameters

A simple model of acute myeloblastic leukaemia (AML) development is introduced, explicitly including cell growth, cell differentiation and cell-cell interaction. Each of these processes is described by a single model parameter. It is hypothesized that the leukaemic cell is characterized by an alteration of only one of these processes. the kinetic behaviour of the AML system is examined separately for possible alterations of each of the three parameters describing the three processes involved. It is shown that, on the basis of the existing data on AML kinetics, the alteration of the growth and cell-cell interaction parameters can be eliminated as a possible source of AML. Thus kinetic data support the modification of the differentiation process as the origin of the AML state. Further, the growth characteristics of normal and leukaemic cells in the presence of each other are analysed. It is shown how the initial growth of leukaemic cells depends on the difference in the differentiation of normal and leukaemic cells and how the same difference determines the decay of normal cells in the presence of the predominantly leukaemic population. Correlations between the kinetic parameters of the normal and leukaemic populations are suggested to characterize the leukaemic state.     

Influence of cryopreservation on human leukaemic clonogenic cells (CFU-L).

The biological and clinical significance of growth characteristics of leukaemic clonogenic cells (CFU-L) cultured from patients has been the subject of many studies. While some investigators collect leukaemic cells in large numbers from blood of untreated patients and store them in a frozen state before use in experiment, others study fresh cells. Since cryopreservation may alter the proliferation and differentiation of CFU-L, we have followed its influence and that of DMSO, used as protective agent and known to be an inducer of differentiation in leukaemic blasts, on the clonogenicity of peripheral blast cells from patients with AML and CML in blast crisis. Our data show that a short incubation with 7.5% DMSO (with or without cryopreservation) induced increase in the clonogenicity and proliferation rate of CFU-L (without morphological changes). The possible causes of these effects as well as the question of aggressivity of leukaemic blasts after the short incubation with 7.5% DMSO are discussed.     

Cell surface properties of L1210 leukaemia cells.

The surface properties of vincristine-colchicine sensitive and resistant L1210 leukaemic cells have been studied using concanavalin A mediated agglutination assay as well as electron microscopic visualization of concanavalin A receptors. 3H-colchicine uptake by the sensitive and resistant lines has also been compared. The resistant L1210 leukaemic cells proved less agglutinable than the sensitive ones at the same concanavalin A concentration. Previous treatments with either colchicine, vincristine or chlorpromazine caused a marked decrease in the agglutinability of the sensitive L1210 leukaemic cells, while agglutination of the resistant ones was lowered slightly by the same treatments. The 3H-colchicine uptake of the sensitive cells was three times higher than that of the resistant ones.     

CFU-C YIELDS FROM THE HAEMOPOIETIC TISSUES OF NORMAL AND LEUKAEMIC RFM MICE

Spleen and bone marrow cells from normal and leukaemic RFM mice have been assayed for numbers of colony forming cells in soft agar (CFU-C). The fluctuations in CFU-C yield observed during the development of myeloid leukaemia are similar to the results from in vitro experiments set up to test a model, and are not incompatible with the idea that interaction between normal and leukaemic cells may modify the yield of CFU-C under the present conditions of culture. Colonies grown from leukaemic spleen and bone marrow cells appear to be derived from the residual population of normal haemopoietic cells within the leukaemic mouse.     

Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin.

A panel of lectins was used to analyse glycoproteins of normal granulocytes and leukaemic myeloid cells. The glycoproteins of detergent-solubilized whole cells were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their lectin-binding properties determined by incubation of the fixed gels with radioiodinated lectins. Normal granulocytes and leukaemic myeloid cells in different stages of maturation possess a cell-surface sialic acid-rich glycoprotein of apparent mol.wt. 115 000 (GP115), that can be labelled by both the lactoperoxidase and periodate/NaB3H4 cell-surface labelling techniques. The sialoglycoprotein of leukaemic myeloblasts has a slightly lower apparent mol.wt., 112000 (GP112). After neuraminidase treatment before cell solubilization, both GP115 and GP112 bind the lectins from Arachis hypogaea (peanut) and Helix pomatia (snail) and have an increased apparent molecular weight of 125000. Two concanavalin A-binding glycoproteins of apparent mol.wts. 98000 and 90000 are present in leukaemic myeloblasts. Concanavalin A binding to these glycoproteins is decreased in more mature leukaemic cells and absent in granulocytes. As concanavalin A binding decreases in the maturer forms, there is a concomitant increase in the binding of Ricinus communis (castor bean) and Maclura aurantiaca (osage orange) lectins to these glycoproteins. Whole granulocytes, but not leukaemic myeloblasts, contain a major cell-surface concanavalin A binding glycoprotein of apparent mol.wt. 130000, which is labelled by the periodate/NaB3H4 technique. Concanavalin A binding to this glycoprotein increases as the morphology of leukaemic cells approaches that of mature granulocytes.     

Acute lymphoblastic leukemia: a study of 25 cases by scanning electron microscopy.

Cells from 25 cases of acute lymphoblastic leukemia (ALL) were studied under the scanning electron microscope (SEM). In 24 of the cases, the vast majority of circulating leukaemic cells had few microvilli. Villous cells were rarely encountered and prominent ridge-like profiles and ruffled membranes were not seen. Only six cases were studied by immunological techniques and four of the cases were of the null type while in two the cells bore detectable T-markers. It seems that ALL is almost always associated with the presence of cells with few microvilli in the peripheral circulation, differing in this respect from most cases of CLL. Although circulating leukaemic lymphocytes with few microvilli are sometimes seen in CLL, the most frequent cell type encountered is a more villous lymphocyte. Differences between leukaemic cells from patients with ALL, CLL and non-lymphoblastic leukaemias are discussed. It appears that SEM may help to distinguish lymphoblastic and nonlymphoblastic leukaemic cells in many instances and can be used as a useful adjunct to other modes of microscopy in the diagnosis of acute leukaemia.     

Differences in electrophoretic mobility and cytochemistry of leukemic T-cells with identical immunologic phenotype (CD1 positive)

Leukaemic cells of acute T-lymphoblastic leukaemia and T-lymphoblastic lymphoma with secondary leukaemic course of disease which showed the same immunological phenotype (cortical thymocytes, thymocyte stage II), could be further differentiated according to their cytochemical pattern and electrophoretic mobility (EPM). Leukaemic cells of T-ALL usually reveal a high EPM, whereas EPM of leukaemic cells of T-lymphoblastic lymphoma was significantly lowered. In cytochemical respect acid phosphatase was positive in both cases of leukaemia. An activity of acid esterase, however, could only be demonstrated in leukaemic cells of T-lymphoblastic lymphoma. The findings are interpreted as manifestation of a different stage of maturity of both T-cell clones with the same immunological phenotype.     

The Wellcome Foundation lecture, 1986. The molecular regulators of normal and leukaemic blood cells

The development of a cell-culture system for the cloning and clonal differentiation of different types of blood cell has made it possible to identify: (i), the proteins that regulate growth and differentiation of different cell lineages in normal and leukaemic blood cells; (ii), the molecular basis of normal and abnormal control of cell development in blood-forming tissue; and (iii), how to suppress malignancy in leukaemic cells. By using myeloid blood cells as a model system, it has been shown that normal blood cells require different proteins to induce cell viability and multiplication (growth-inducers) and differentiation (differentiation-inducers), that there is a hierarchy of growth-inducers which act at various stages of cell development, and that a growth-inducer can switch on production of a differentiation-inducer. Gene cloning has established a multigene family for these proteins. Identification of these proteins and their interaction has shown how growth and differentiation are regulated in normal development and demonstrated the mechanisms that uncouple growth and differentiation so as to produce malignant cells. Normal cells require an external source of growth-inducing protein for cell viability and multiplication. Cells can become leukaemic by genetically changing this normal requirement for growth without blocking response to normal differentiation-inducers. The mature cells induced by adding these normal protein-inducers are then no longer malignant. Other genetic changes which inhibit differentiation by the normal blood-cell regulatory proteins can occur in the evolution of leukaemia. But even these leukaemic cells may still be induced to differentiate by other compounds that can induce differentiation by alternative pathways. The differentiation of leukaemic to mature cells, which stops the cells from multiplying, results in the suppression of malignancy by bypassing genetic changes that produce the malignant phenotype. The activity of blood-cell growth- and differentiation-inducing proteins has been shown in culture and in the body. They can, therefore, be clinically useful to correct defects in the development of normal and leukaemic blood cells.     

Autologous responses to human leukaemic cells in mixed leucocyte culture

Summary Cryopreserved leukaemic blasts and remission non-T cells from 22 patients with acute leukaemia (15 lymphocytic, 7 non-lymphocytic) were tested as stimulators of autologous remission T cells and normal allogeneic T cells in primary and secondary MLC. In most cases the autologous response elicited by leukaemic cells was less than or equal to that elicited by remission non-T cells. However, T cells from 2 patients in long-standing first remission from ANLL displayed greater proliferation in response to leukaemic blasts than to remission non-T cells in both primary and secondary MLC. The results are suggestive of sensitization of these 2 patients to leukaemia-specific antigens, but other possible explanations are discussed. Abbreviations used: MLC, mixed leucocyte culture; ANLL, acute non-lymphocytic leukaemia; ALL, acute lymphoblastic leukaemia; AMLR, autologous mixed lymphocyte reaction; NK cells, natural killer cells; MNC, mononuclear cells     

In vitro proliferation of normal and leukaemic human leukocytes controlled by an inhibitory endopeptide

GI-3, an endogenous inhibitory fraction isolated from leukocytes, selectively inhibits the proliferation of granuloid precursor cells in a non-toxic manner. Its active principle was determined as an acidic chlor-tolidine positive decapeptide [ 3 ]. The in vitro effect on normal and acute leukaemic human bone marrow and blood cells was examined. A dose dependent inhibition by GI-3 of 3H-TdR incorporation into myeloid cells of normal bone marrow was found, the sensitivity of human cells being higher than that of rat cells. The proliferation of the target leukaemic bone marrow and blood cells (AML, AMMoL) was also decreased by the endogenous inhibitor in a dose dependent manner in untreated subjects as well as in patients in remission or relapse. The rate of inhibition of leukaemic of well-known cytostatics (adriamycin hydrochloride, dianhydrogalactitol) applied for comparison. Beyond its direct cytostatic effect, GI-3 could be used in the differential diagnosis of blastic leukaemias, complementing the routine cytochemical methods.     

Preferential induction of apoptosis of leukaemic cells by farnesol

Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 microM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.     

Generation time of leukaemic blast progenitor cells

Previous studies have indicated that the generation time of human leukaemic cells is longer than that of normal haematopoietic cells. We have employed a modification of the thymidine (TdR)-suicide technique to measure directly the generation time of leukaemic progenitor cells capable of colony formation. The results obtained with two human leukaemic cell lines (KG-1 and HL-60) and with blast progenitor cells from two patients with acute myelogenous leukaemia indicate generation times ranging from 9 X 0-22 X 0 hr and S-phase durations ranging from 5 X 5-8 X 0 hr. Using the same technique, the generation time of normal bone marrow CFU-c was determined to be 9-11 hr. These findings suggest that the proliferation rate of human leukaemic blast progenitor cells is similar to that of normal haematopoietic stem cells.     

Higher tyrosine protein kinase activity in resting lymphocytes than in proliferating normal or leukaemic blood cells

We have studied tyrosine phosphorylation in particulate fractions from 11 leukaemic cell lines by using as substrate either a synthetic tyrosine containing peptide or the endogenous proteins. The results were compared with those obtained using similar fractions from normal lymphocytes and bone marrow cells. Particulate fractions from all the leukaemic cell lines and normal bone marrow cells exhibited lower levels of tyrosine protein kinase activity compared to normal lymphocytes. When the phosphorylation of endogenous substrates was assayed, proteins were phosphorylated on tyrosine residues (rather than serine or threonine residues) to a larger extent in normal lymphocytes than in leukaemic cell lines. Separation of labelled endogenous substrates on sodium dodecyl sulfate-polyacrylamide gels showed a number of phosphorylated alkali-resistant bands in the range 14-175kd in the lymphoid cell lines; normal lymphocytes exhibited a smaller number of strongly phosphorylated bands. Normal lymphocytes from different individuals showed reproducible patterns of phosphorylated substrates. Normal bone marrow cells and myeloid leukaemia lines showed weak, if any, phosphorylation. Among the leukaemic cell lines no particular pattern of phosphorylated substrates common to cells of similar phenotype could be detected. We suggest that the level of overall tyrosine protein kinase activity in these fractions reflects their position in the cell cycle rather than their normal or malignant status.     

FUNCTIONAL CELL COMPARTMENTS IN A RAT MODEL FOR HUMAN ACUTE MYELOCYTIC LEUKAEMIA

Functional cell compartments were studied in a rat model for human acute myelocytic leukaemia (AML). This was done by tracing the distribution of injected ⁵¹Chromium-labelled leukaemic cells in the body. It was concluded that two functional compartments can be distinguished in acute leukaemia, i.e., a rapidly exchangeable pool of cells (including the circulating blood pool, the marginal noncirculating blood pool and the rapidly exchangeable tissue pool; RETP) and a slowly exchangeable tissue pool (SETP). The sizes of these various compartments were roughly quantified at various stages of the disease by calculations based on the principle of isotope dilution and organ weight measurements. As the leukaemia progresses, the size of the SETP increases significantly relative to the size of the RETP. Simultaneously, the exchange rates of leukaemic cells between the organs and the blood decrease. The blood transit time of leukaemic cells was also significantly prolonged, as is the case in human AML.     

Na+/H+ exchanger 1 inhibition contributes to K562 leukaemic cell differentiation

The effect of hypoxia on the differentiation of chronic myeloid leukaemic K562 cells were studied, as was the role of the NHE1 (Na+/H+ exchanger 1). Hypoxia induced differentiation of K562 cells as seen by modifications in their morphological features, up-regulation of C/EBPα (CCAAT/enhancer-binding protein α), and marked IL-8 (interleukin-8) release. Inhibition of NHE1 under hypoxia additionally enhanced the level of C/EBPα and further promoted leukaemic cells differentiation. Pharmacological inhibition of p38 MAPK (mitogen-activated protein kinase) also significantly suppressed C/EBPα expression under hypoxia conditions after NHE1 inhibition. These results indicate the enhancement of hypoxia-induced K562 differentiation by NHE1 inhibition, which may be due to up-regulation of C/EBPα via p38 MAPK signalling pathway, which suggests a possible therapeutic target of NHE1 under hypoxia microenvironment in the treatment of leukaemic diseases.     

Functional cell compartments in a rat model for human acute myelocytic leukaemia.

Functional cell compartments were studied in a rat model for human acute myelocytic leukaemia (AML). This was done by tracing the distribution of injected 51Chromium-labelled leukaemic cells in the body. It was concluded that two functional compartments can be distinguished in acute leukaemia, i.e., a rapidly exchangeable pool of cells (including the circulating blood pool, the marginal noncirculating blood pool and the rapidly exchangeable tissue pool; RETP) and a slowly exchangeable tissue pool (SETP). The sizes of these various compartments were roughly quantified at various stages of the disease by calculations based on the principle of isotope dilution and organ weight measurements. As the leukaemia progresses, the size of the SETP increased significantly relative to the size of the RETP. Simultaneously, the exchange rates of leukaemic cells between the organs and the blood decrease. The blood transit time of leukaemic cells was also significantly prolonged, as is the case in human AML.     

Study of cell death in Friend leukaemia.

Cell death of splenic Friend leukaemic cells has been studied in vivo, using 125I-UdR and 3H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of 3H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8% of the splenic cellular DNA being renewed per day. Those results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.