Human CML Research Articles

Induction of cellular differentiation and apoptosis by signal transduction inhibitors

We have isolated signal transduction inhibitors of low molecular weight from microorganisms and plants. Since inducers of differentiation and apoptosis may be developed as new anticancer agents, we have studied induction of differentiation and apoptosis in neoplastic cells by our signal transduction inhibitors. Aristeromycin isolated as an Abl function inhibitor induced erythroid differentiation in human CML K562 cells. Aristeromycin may induce differentiation by inhibition of methylating reactions in the cell. We isolated dephostatin from Streptomyces as a tyrosine phosphatase inhibitor, and synthesized its stable analogue, 3,4-dephostatin. The stable analogue, 3,4-dephostatin, potentiated NGF-induced morphological differentiation in rat pheochromocytoma PC12h cells, possibly by inhibition of tyrosine dephosphorylation of MAPK. Erbstatin, a tyrosine kinase inhibitor, induced morphological apoptosis and internucleosomal DNA fragmentation in mouse leukemia L1210 and human SCLC cells. Erbstatin was shown to induce apoptosis by hydrogen peroxide formation. Thus, these signal transduction inhibitors appear to be useful tools for the mechanistic study of cellular differentiation and apoptosis.

HCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules

A 19 kDa protein was identified in specific granules of human neutrophils. A full-length cDNA clone was isolated from a human CML cDNA library, based on amino-acid sequences of isolated tryptic fragments. This clone includes the recently identified cDNA for FALL-39/CAP-18. Aminoacid sequences of proteolytic fragments derived both from the conserved N-terminal cathelin-like region and the highly variable C-terminal region characteristic of this family of bactericidal, LPS binding proteins, were in complete agreement with the sequence deduced from the cDNA. Thus, the 19 kDa protein is hCAP-18, stored as a ‘pro-peptide’ in specific granules.

Differential induction of adult and fetal globin gene expression in the human CML cell subline KU-812F/33.

Various chemicals which are known to have positive effects on differentiation of some erythroid cell lines were tested on a human chronic myelogenous leukemia cell line, KU-812F. Succinic acid, 5-azacytidine, daunomycin, and hemin showed a positive effect. Among them, hemin and 5-azacytidine were the most effective inducers for erythroid differentiation of KU-812F cells. Dimethylsulfoxide, cytosine arabinofuranoside, and sodium n-butyrate showed no effect. In addition, subclone KU-812F/33 derived from the KU-812F cell line showed differential expression of the beta- and gamma-globin genes in the presence of either 2 microM 5-azacytidine or 40 microM hemin. Hemoglobin synthesis in differentiated KU-812F/33 cells was analyzed by isoelectric focusing gel electrophoresis, and S1 mapping analysis of beta- and gamma-globin mRNA was performed. After treatment with 5-azacytidine, the beta-globin gene expression was predominantly enhanced (18.75-fold higher level of beta-globin mRNA). After treatment with hemin, the most notable increase was in the gamma-globin gene expression (1.83-fold higher level of gamma-globin mRNA), while no increment of beta-globin was observed.

The origin and spread of human leukemia.

The human leukemias are a group of hematologic neoplasms characterized by uncontrolled proliferation of cells concerned with blood cell production. The cause(s) of human leukemia remains unknown. Bone marrow (BM) is believed to be the site of origin of human leukemias, although the specific locus(i) and/or cell(s) from which it arises have not been definitively identified. Generally, human leukemias and related proliferative diseases are thought to be clonal in nature; affecting a single hematopoietic stem cell, which then proliferates and replaces the marrow of normal hematopoietic stem cell systems. The condition is believed to be malignant in nature. Results of our current morphologic studies on well-fixed, ideally-stained thin sections of plastic-embedded bone marrow biopsies (BMB) from a large number of acute (AML, ALL) and chronic (CGL, CLL) leukemia patients suggest that human leukemias may not be clonal diseases. Instead, a large population of other resident cells--'endosteal cells'--appears to become involved in the process and it is possible that all members of this group enter the activity simultaneously. This change (transformation) in the endosteal cell population might be due to an abnormality (qualitative or quantitative) of diffusable, humoral factors (yet to be identified) that are responsible for the growth and proliferation of these hematopoietic precursor cells. In this context, the human leukemias may be considered not as malignant, but rather the result of an aberration of factor(s) that control hematopoiesis. In this respect, the human leukemias, particularly AML, ALL and CML, might be analogous to pernicious anemia (megaloblastic anemia) as it was understood 40-50 years ago.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobin Synthesis of Both Adult and Fetal Types in a Human CML Cell Line1

We present evidence for the continuous erythroid differentiation of a cell line, KU-812-F, without the addition of an inducer. Erythroid differentiation of these cells was confirmed according to the following findings: 1) erythroid morphology; 2) existence of glycophorin A and carbonic anhydrase I as a membrane marker protein and an enzyme characteristic of erythroid cells, respectively; 3) synthesis of adult type (HbA and HbA2) and fetal type (HbF) hemoglobins, as detected on isoelectric focusing; and 4) transcription of the alpha-, beta-, and gamma-globin genes, as detected on Northern blot analysis.

Unique monoclonal antibodies against soluble membrane proteins of human CML myelocytes reactive with human myeloid progenitor cells.

Monoclonal antibodies of IgG1 type immunoglobulin, directed against soluble CML antigen isolated from the reactive CML peripheral myelocytes, were reported. These MAbs were further investigated for their reactivity by 125I-Protein-A binding assays, indirect immunofluorescence tests, cytotoxicity, SDS-PAGE, immunoelectrophoresis, and by immunodiffusion suggesting that they recognized antigens specific mostly to undifferentiated cells. These were tested against various leukemic peripheral blood leukocytes, bone marrow cells, established cell lines of various origin, and with many solid tumor cells and demonstrated specific reactivity with CML myelocytes alone and cell lines of myeloid origin. Indirect immunoperoxidase staining of single cell preparation revealed peroxidase localization in most promyelocytes and in few mature myelocytes from CML PBL/BM cells, thus helping in identifying the exact type (morphology) involved in reacting specifically with these MAbs.

Characterization of an antigen expressed by human natural killer cells.

A monoclonal antibody, anti-N901, was produced by fusing NS-1 myeloma cells with spleen cells of a mouse immunized with human CML cells. This antibody was reactive with a subpopulation of peripheral blood LGL, including the natural killer cells. Monocytes, granulocytes, B cells, T cells (T3+ cells), erythrocytes, and platelets were nonreactive. The N901-positive cells in the peripheral blood were heterogeneous with respect to expression of other cell surface antigens. The majority of N901+ cells co-expressed T11, Mo1, and HNK-1, whereas a smaller percentage expressed T8. Ia, T3, T4, Mo2, or B1 antigens were very uncommon on N901+ cells. The heterogeneity of the N901+ LGL was further investigated by examining the expression of N901 antigen on a series of cloned normal human NK cell lines. N901 antigen was expressed by each of the NK cell lines tested, and by a minority of cloned T cell lines without NK activity. Anti-N901 does not block NK activity and can be used to rapidly purify functional NK cells for further study.

Identification of human CML target. HLA-B locus (B12) antigen variants defined by CTL generated between B locus-identical (B12) responder-stimulator pairs.

The genetics of human CML targets were studied by seven CTL generated in combinations iun which the responder/stimulator difference was limited to one (or two) HLA-A, -B, or -C antigens. Unstimulated peripheral blood lymphocytes were used as targets. CTL sensitized against antigens A11, Aw31, or B17 lysed all cells bearing the respective target from a large panel of cells from unrelated individuals. Hence, at least one CTL clone was directed against the HLA antigen molecule. However, all CTL also exerted cross-kill to cells not sharing the stimulating HLA antigen. For two CTL, the target of the cross-kill was not clarified. Five CTL were found where the cross-kill was directed against antigen HLA-B12 (Bw44 and Bw45). All these cTL were generated in R/S pairs identical for B locus antigens (Bw44/Bw35 heterozygotes). The individual CTL lysed different parts of the panel of B12-positive target cells. The interpretation is that these CTL detect subtypes of HLA antigens, but alternative possibilities are also considered. Four B12 subtypes are described, tentatively designated as B12-related CML targets. Identification of HLA-B-related CML targets represent CML "typing" of HLA-antigen differences that were not detected serologically. The subtypes can now be tested for their possible functional significance.

Blast crisis associated with granulocytic leukemia in strain 13 guinea pigs

A transplantable granulocytic leukemia (GL-13) in inbred strain 13 guinea pigs had been shown previously to have many characteristics in common with human CML except that a blast crisis did not occur in the terminal stage of the disease. We report here that, after the 25th transplant generation, leukemic guinea pigs began to develop a blast crisis similar to that seen in human CML. This feature of the disease is still present after 90 transplant generations. The transplantation conditions necessary to produce the leukemia (now referred to as GL-13-BC) with a predictable clinical and hematological course over a short interval are described. Thus, s.c. injection of 3.0 × 10 6 leukemic cells results in a slowly rising leukocyte count, due mainly to increasing numbers of myelocytes and mature granulocytes, at about 3 weeks after injection. At this early stage the spleen weights of the leukemic animals are about seven times greater than those of normal controls. Approximately 4–7 days later the leukocyte count rises sharply due to a blast crisis and the animals die shortly thereafter with a mean survival time of 30 (range 25–32) days. It is proposed that the GL-13-BC leukemia could serve as a useful model for investigating the problem of blast crisis associated with human CML.

Cytotoxic T lymphocytes directed against HLA-Bw35-linked target determinants show differences in sensitivity toward antibiotics during sensitization period

Recently, we demonstrated that the outcome of human CML ∗ was influenced by the presence of antibiotics in the culture medium. In earlier studies we had found that cytotoxic T lymphocytes could recognize HLA-Bw35-linked target determinants. These so-called Bw35 a and b determinants showed Mendelian segregation. We describe in this article a difference between the Bw35 a and b cytotoxic T cells in their sensitivity toward antibiotics. The lysis against the Bw35 b cytotoxic determinant was not influenced by either the presence or the absence of antibiotics during the sensitization period. whereas the lysis against the Bw35 a cytotoxic determinant was drastically diminished when the effector cells were cultured in the absence of antibiotics during the sensitization period.

Chemotherapy of autochthonous myeloid leukemias (Chloroleukemias) in Wistar rats.

After injection of 15 mg/kg ethylnitrosourea (ENU) weekly for 15 weeks to adult male Wistar rats (total dose: 225 mg/kg) about 10% of the animals developed myeloid leukemias (chloroleukemias), which resemble the chronic myeloid leukemia in man (CML) (peripheral blood picture, tissue infiltration, chronic course as compared to immature-cell rat leukemias). Monotherapy with busulfan effected no remissions. The median survival time after daily treatment with busulfan was 29.5 days (range: 7-70); it was significantly shorter than that of untreated controls (median: 47.5 days, range: 22-81). After weekly application of 20 mg/kg and 50 mg/kg Cyclophosphamide the median survival time increased to 69.5 (range: 26-114) and 61.5 (range: 20-92) days, respectively. Rate and duration of remissions after repeated weekly single doses of cyclophosphamide were positively correlated with the increase in single dose; the high dose-intermittent treatment with 50 mg/kg CPA/week yielded complete remissions in all treated animals. Despite these remissions however, no significant increase in survival time could be observed in comparison with untreated controls. The comparability of autochthonous chloroleukemias in the rat with human CML is discussed.

Patterns of antibody reactivity against selected human leukemia cell lines.

Absorption procedures which allow the production of a selectively cytotoxic anti-human lymphocyte serum are described. Although the production of a reagent whose reactivity is restricted. Although the production of a reagent whose reactivity is restricted exclusively to lymphocytes may be achieved by exhaustive absorption steps using fresh human erythrocytes, CML cells, and fetal liver cells, a more realistic alternative is the use of appropriately selected cultured human leukemia cell lines. Data are presented which show how these cell lines may be employed to selectively manipulate the cross-reactivity spectrum of ALS. Pre-treatment of donor bone marrow cells prior to transplantation with a selectively lymphocytotoxic ALS has been shown to allow transplantation of bone marrow across major histocompatibility barriers in rodents without the occurrence of GvH reactions, and it is the purpose of the present investigations to show that an analogous anti-human ALS can be prepared which possesses the required degree of selectivity to allow its application for human bone marrow transplantation.

The preparation and cytotoxic properties of211at labelled concanavalin a bound to cell membranes

A method of labelling biologically active proteins with the alpha emmitting halogen211At is presented. The labelling procedure is discussed with reference to the chemistry of astatine. Proteins which have been labelled retain approximately 50% of their original biological activity. Using cell specific labelled proteins, dose response curves are given indicating that such reagents are extremely cytotoxic, D37 human CML cells=5 atmos211At/cell. The research potential of211At labelled biologically active proteins is briefly discussed.