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Here, a new acute myelomonocytic leukemia (AMML) cell line, JIH-3, is reported, and its biological characteristics are described. JIH-3 cells were maintained without any cytokines for 27 months. The JIH-3 cell line showed typical myelomonocytic morphological features. Additionally, it mainly expressed myeloid and monocytic markers (CD13, CD14, CD11b, CD15 and CD33), although it also expressed other antigens such as the markers of T and B lymphocytic lineage as well as stem cell, progenitor cell, and natural killer cell-related antigens (CD4, CD5, CD7, CD10, CD22, CD34, CD38, HLADR, CD16/CD56 and CD56); the expression of these markers, suggested that this cell line was in the early stage of myelomonocytic differentiation. Cytogenetic analysis initially showed a karyotype of 46, XY, del(7) (p1?3p2?2). During the passage period, the cells with this karyotype gradually decreased and were replaced by cells with a 45,XY,dic(4;7)(p11;p11),del(15)(q2?2) karyotype. Chromosome painting showed a deletion in the short arm of chromosome 7 for del(7)(p1?3p2?2) and der(4;7)(p11;p11). The latter had larger deleted segment than the former. Fluorescence in situ hybridization (FISH) revealed the dicentric nature of der(4;7), and Multiplex FISH (M-FISH) confirmed that der(4;7) was an unbalanced translocation. A deletion involving the 7p region on dic(4;7)(p11;p11) harbors many genes, including CDC2L5, C7ORF11, C7ORF10 and INHBA. Haploinsufficiency of the genes on 4p, 7p and 15q caused by deletions of 4p, 7p and 15q2?2 that resulted from dic(4;7)(p11;p11) and del(15)(q2?2) may play important roles in leukemogenesis and in the establishment of the JIH-3 cell line. JIH-3 cells did not express multidrug resistance (MDR)-related genes and apoptosis-related genes such as MDR1, multidrug resistance-related protein, lung resistance protein, BCL-2, Bax, GS-π or Fax, only P21 expression was detected, which probably leads the MDR indirectly through inhibition of the activities of cyclin-dependent kinase (CDK). JIH-3 cells had tumorigenic capacity in nude mice. In conclusion, JIH-3 is a new acute myelomonocytic leukemia cell line. It is from a well-characterized background and provides a new useful tool for the study of leukemia patients with a 7p deletion., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

CCAAT/enhancer binding protein alpha (C/EBPα) induction induces monocytic differentiation even in acute myeloid leukaemia (AML). In this study, the induction/activation of C/EBPα in myelomonocytic AML was investigated using a combination of all-trans retinoic acid (ATRA) and RAD001 (Everolimus), a mammalian target of rapamycin complex 1 (mTORC1) inhibitor. Combining these agents increased PU.1, C/EBPε and C/EBPα expression, increased the p42/p30 C/EBPα ratio, and decreased C/EBPα phosphorylation at serine 21, and was accompanied by growth inhibition, induction of CD11b expression and apoptosis in AML cell lines. Thus, agents that induce sufficient levels of C/EBPα expression might be useful in treating AML., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We found recombinant human soluble thrombomodulin (rTM) induced growth arrest and differentiation of THP-1 cells by activating JNK/c-Jun signaling. Further activation of JNK by 1,25-(OH)(2)D(3) significantly enhanced rTM-mediated growth arrest and differentiation of THP-1 cells. Importantly, forced expression of domains 1, 2 and 3 of TM (TMD123) induced growth arrest and differentiation of leukemia cells freshly isolated from individuals with AMLs of M4/M5-French-American-British classification subtypes, but not those with less advanced AML. Further studies indicated that the epidermal growth factor-like domain of TM was critical for the anti-leukemia effects of TM and these effects were independent of protein C activation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Quantum dots (QDs) are nanometer scale fluorescent semiconductors that are increasingly used as labeling tools in biological research. These nanoparticles have physical properties, such as high quantum yield and resistance to photobleaching, that make them attractive molecular probes for tracking hematologic cells. Here, we show that QDs attached to a transporter protein effectively label all hematologic cells tested, including cell lines and malignant and non-malignant patient samples. We demonstrate that dividing cells can be tracked through at least four cell divisions. In leukemic cell lines, some cells remain labeled for 2 weeks. We show that QDs can be used to follow cells as they differentiate. QDs are seen in monocyte-like and neutrophil-like progeny of labeled HL-60 myeloblasts exposed to Vitamin D analogues and DMSO, respectively. QDs are also observed in monocytes generated from labeled CD34+ cells. In addition, QDs attached to streptavidin can target cells with differing cell surface markers, including CD33. In summary, QDs have the ability to bind to specific cells of interest, be taken up by a diverse range of hematologic cells, and followed through many divisions and through differentiation. These results establish QDs as extremely useful molecular imaging tools for the study of hematologic cells.

We report a patient with juvenile myelomonocytic leukemia who had two cytogenetically independent clones at the time of diagnosis. Fluorescence in situ hybridization analyses showed that 42.5% of myeloperoxidase(+) cells and 27.3% of CD20(+) cells had three signals for chromosome 8, while 13.1% of myeloperoxidase(+) cells and 6.0% of CD20(+) cells had del (Y). However, a great majority of CD3(+) cells had no numerical aberration of the two chromosomes. The two karyotypically abnormal clones might have developed from multipotential hematopoietic stem cells with the potential to differentiate into myeloid and B-lymphoid lineages, but not T-lymphoid lineage.

Analyzing the regenerative compartment in the blast cell population of patients with acute myeloid leukemia (AML) may yield important insights into the mechanisms of disease progression. Here we present findings with a human AML cell line (AML-SP1), initiated from leukemic precursor cells and consecutively propagated by serial xenotransplantation in vivo. AML-SP1 maintained the characteristics of a human AML, consistently exhibiting a small leukemic side population (SP) of blast cells with high Hoechst 33342 exclusion. In the AML-SP1 line, an increased expression of the ABC transporters MDR1, MRP, ABCG2 and ABCA3 was found in the SP cells. The detection of ABCA3 in leukemic progenitor cells merits further investigation with regard to intracellular drug transport in AML blast cells. In vivo propagation of leukemias, such as AML-SP1 is a model system of maintaining the populational heterogeneity of AML disease, especially the unique characteristics of leukemic SP cells.

The ability of leukemic cells to differentiate to mature dendritic cells (DCs) was investigated in six acute myelomonocytic or monocytic leukemia cases. It was found that CD14 positive cells were more efficiently changed to CD83 positive mature typed DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) compared with CD14 negative cells. Such leukemia derived DCs expressed a sufficient level of costimulatory molecules (CD80 and CD86), and were shown to be monoclonal based on an the X-inactivation analysis. They also stimulated not only allo- but auto-T lymphocytes, which thereafter became cytotoxic T lymphocytes (CTLs).

We have investigated the protein expression of the chemokine monocyte chemotactic/chemoattractant protein-1 (MCP-1) in various human myelomonocytic leukemia cell lines. Applying specific ELISA, we demonstrated that this chemokine is produced constitutively by the cell lines HL-60, ML-2, MONO-MAC-6 and MUTZ-3 ranging between 440 and 1400 pg/ml MCP-1 per million cells. In the culture medium of two other unstimulated cell lines, MONO-MAC-1 and THP-1, almost no MCP-1 was detected. Stimulation of HL-60 and MONO-MAC-6 with lipopolysaccharide (LPS), and stimulation of ML-2 and MUTZ-3 with 12-tetradecanoyl phorbol 13-acetate (TPA) dramatically increased the MCP-1 level in the culture medium. The highest amount of MCP-1 (> 80 ng/ml within 24 h) was achieved by TPA stimulation of MUTZ-3 cells. Out of 15 cytokines tested for induction or enhancement of MCP-1 secretion, interleukin-3 (IL-3), IL-6, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and tumor necrosis factor (TNFalpha) were able to augment (twofold to 12-fold) the MCP-1 level in the culture medium of MONO-MAC-6 cells. While the antinflammatory cytokines IL-4, IL-10 and IL-13 failed to suppress MCP-1 secretion, the glucocorticoid dexamethasone strongly inhibited the MCP-1 production of unstimulated and stimulated MONO-MAC-6 cells. Thus, several regulatory elements are involved in MCP-1 secretion. Despite the quantitative differences of MCP-1 production among the cell lines analyzed, our results demonstrated a constitutive secretion in differentiation-arrested myelomonocytic leukemia cell lines and emphasize the usefulness of these malignant cell lines as models to study MCP-1 secretion and regulation.

A case of acute monocytic leukemia (AMoL) with t(5;11)(q31;q23) and a case of acute myelomonocytic leukemia (AMMoL) with t(5;11)(q35;q13.1) are reported. The translocation between the long arm of chromosome 11q and that of chromosome 5q with leukemia have been rarely reported. Though breakpoint of both cases were subtlety different, they had morphologically monocytic character and showed hyperleukocytosis and chemoresistance.

The biphenotypic cell line BW-90 was established from the peripheral blood of a a patient with a refractory acute myelomonocytic leukemia. All cells were HLADr+, CD34-. Dual color flow cytometry showed simultaneous expression of myeloid (CD33) and B-lymphoid surface markers (CD19) on 60% of cells, CD54 on 91% of cells. Lymphoid lineage markers included CD20/CD22 coexpressed on 89% of cells, CD71 (70%), CD11a (48%), CD18 (54%), and surface lambda light chain (33%). Exposure to various cytokines individually and in combination for up to 14 days had no effect on cell proliferation or differentiation. Only long-term (10-14 days) exposure to 5637 cell-conditioned medium (CCM) induced growth inhibition and differentiation along the monocytic pathway. Differentiation-inducing agents retinoic acid (RA), dimethyl sulfoxide (DMSO) and phorbol 12-myristate 13-acetate (PMA) did not induce differentiation. Differentiation into the monocytic pathway was induced by 5-azacytidine (5AzaC) alone or in combination with verapamil (VP). The BW-90 cell line may serve as a model to study early steps of leukemogenesis and early hematopoiesis. It may provide insight leading to development of an effective therapy for treatment-resistant biphenotypic leukemias.

In 141 adult patients with diagnosis of acute myeloid leukemia the overall expression and intensity of expression of CD34 antigen on leukemic cells was investigated. Myeloid blasts were tested by applying direct immunofluorescence staining using anti-CD34 fluorescein monoclonal antibody in flow cytometry. CD34 antigen was found in 73 out of 141 (51%) cases and in particular in M0, M1 and M4 French-American-British (FAB) cytotypes, while M3 and M5 cases were rarely positive. In patients whose blasts expressed CD34 antigen a significantly lower rate of complete remission (CR) was observed as opposed to CD34 negative cases (61% vs 88%) (P = 0.001). Furthermore, a negative correlation between high intensity of CD34 expression, measured as a mean fluorescence index (MFI), and CR rate was observed. In particular, patients with a higher CD34 fluorescence intensity (MFI > 23), showed a further reduction in CR rate (48%). Also, these patients had a significantly lower overall survival (P = 0.03) as compared to patients with no expression of CD34 and patients with CD34 MFI < 23. In conclusion, these findings confirm that CD34 expression is frequently associated with "immature" FAB cytotypes (M0, M1 and M4) and with a reduced probability to achieve CR. Furthermore, a high CD34 intensity of expression should be considered as a reliable poor prognostic factor.

The in vivo induction of the differentiation of murine WEHI-3B D+ myelomonocytic leukemia cells was measured by flow cytometry, simultaneously staining leukemia cells for the marker exogenous beta-galactosidase and for differentiation by the antigen Mac-1 (CD11b/CD18). The WEHI-3B D+ leukemia cells were transfected with the E. coli lac-Z gene by electroporation and subclones that constitutively expressed high levels of the lac-Z gene product beta-galactosidase were established. Flow cytometric analyses of cells in the peritoneal cavities of mice bearing leukemia cells showed that cells continued to express beta-galactosidase for at least 14 days, and they were distinguishable from host-derived cells in vivo by their expression of the transfected gene. Simultaneous determination of the beta-galactosidase activity and Mac-1 content of cells in the peritoneal cavities of mice revealed that administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to the mice enhanced the expression of Mac-1 antigen by beta-galactosidase-positive cells. The results demonstrate that G-CSF may have clinical potential as a therapeutic differentiating agent, and that flow cytometric analysis provides a useful in vivo system to evaluate the therapeutic potential of agents capable of inducing terminal differentiation.

Acute myeloblastic leukemia (AML) with t(8:16) or its variant t(8:V) has been rarely reported. A high proportion of patients are infants and children, often with a bleeding tendency and disseminated intravascular coagulopathy (DIC). Only one-third of the de novo patients remain in the first complete remission following multiagent chemotherapy and bone marrow transplantation (BMT). Morphocytochemically, the disorder is classified as an M5, M4, or M4/M5 variant. In the presented case, with the variant t(8:19)(p11:q13), comprehensive light and electron microscopic blast cell characterization showed monocytic and granulocytic features compatible with the M4 subtype (on the monocytic predominance range of the French-American-British classification scale). Although hemophagocytosis, one of the hallmarks of the disease, was rare in our patient, numerous autophagic vacuoles were present. Immuno- and genotyping showed a myelomonocytic phenotype with no evidence of early progenitor antigen expression or mixed leukemia. These results and those of previous reports support the high specificity of t(8:16) or its variants to the unique M4/M5 type leukemia and the role of a gene on 8p11 in this specific transformation.

In order to demonstrate possible roles of retrodifferentiation in relapses after differentiation therapies, we have established a retrodifferentiated cell line (RD-1) from a single rat myelomonocytic leukemia cell which differentiated into a macrophage-like cell by treatment with lipopolysaccharide (LPS). The established RD-1 cells showed microscopic features slightly maturer than their parent cells. The RD-1 cells had the ability to differentiate into macrophage-like cells by treatment with fewer doses of LPS than those for parent cells. All rats inoculated with the parent cells (more than 10(2)/rat) died within 50 days. Rats inoculated with 10(4) RD-1 cells survived for more than 120 days, whereas two out of four rats inoculated with 10(5) cells and all the rats inoculated with 5 x 10(5) cells died of leukemia. These results suggest that RD-1 cells are retrodifferentiated cells from a single rat myelomonocytic leukemia cell which differentiated into a macrophage-like cell; they have similar phenotypes and lower tumorigenicity than the parent cells and they also suggest that the appearance of retrodifferentiated leukemia cells may be responsible for relapse after differentiation therapy for leukemia in some cases.

We have found that LPS induces the differentiation of an LPS-resistant subline (LR) of rat myelomonocytic leukemia cell line, c-WRT-7, in vivo, which are resistant to the differentiation inducing effects of LPS in vitro. Furthermore, we have found that the differentiation of LR cells induced by LPS is inhibited by superoxide dismutase, which is one of radical scavengers. Accordingly, we have examined the differentiation inducing effects of xanthine oxidase, a potential source of oxygen radicals, on LR cells in vitro and in vivo. Xanthine oxidase induced the differentiation of LR cells into macrophage-like cells in vitro; and superoxide dismutase inhibited the differentiation of LR cells induced by xanthine oxidase both in vitro and in vivo. These results suggest that oxygen radicals are involved in the differentiation inducing effects of LPS.

High dose methylprednisolone (HDMP) has been used in induction treatment and in leukopenia of acute leukemia. We report the case of a 14-year-old girl with acute myelomonocytic leukemia (AMML) who developed a pseudorelapse after receiving an oral HDMP trial (30 mg/kg/day PO in the morning for 15 days) to overcome neutropenia. She presented with severe bone pain at the end of treatment. Bone marrow examination revealed 53% young promyelocytes (some with a large nucleolus), 2% myeloblast, 5% monocyte, and increased cellularity. Bone pain and promyelocytes regressed spontaneously while maintenance treatment continues. We advise that a pseudorelapse must be borne in mind after HDMP and probably GM-CSF treatments in acute leukaemias.

Chondroitin sulfates extracted and purified by different manufacturers were tested to evaluate their effects on proliferation and differentiation processes of U-937 cells. The different chondroitin sulfates were evaluated for purity, structure and physicochemical properties. The three chondroitin sulfates utilized did not present other contaminant glycosaminoglycans and proteins and had about the same relative molecular mass but different disaccharide patterns and charge density. Chondroitin sulfates with small amounts of disulfated disaccharides and low charge density, at 5 micrograms/ml concentration, doubled (about + 133%) cell proliferation in comparison to controls. In contrast, chondroitin sulfates with large amounts of disulfated disaccharides and high sulfate to carboxyl ratio were less effective (about + 15%) in stimulating cell proliferation at low concentration. A decrease of U-937 cell proliferation was observed in proportion to the increased amounts of chondroitin sulfate with low sulfate to carboxyl ratio. On the contrary, chondroitin sulfate with large amounts of disulfated disaccharides produced increased cell proliferation depending on concentration. Small amounts (5-10 micrograms/ml) of chondroitin sulfates with low charge density reduced the differentiative process of U-937 cells. Chondroitin sulfate with large amounts of disulfated disaccharides and high charge density seemed to be able to produce a significant decrease of differentiative processes only at very high concentrations (1000 micrograms/ml). These contrasting effects of chondroitin sulfates with different disaccharide patterns (and structure) and charge density on a leukemia cell line could help to explain the regulation of proliferative and/or differentiative processes of hemopoietic cells. This is underlined by the changes of types, physicochemical properties and structure of glycosaminoglycans induced by different extracellular factors and agents.

The use of lithium chloride in manic-depressive patients and in patients receiving myelo-suppressive cancer chemotherapeutic agents is accompanied by a sustained leukocytosis due to an increase in granulocyte production. This property suggests that lithium chloride may have effects on hematopoietic differentiation. Treatment of cultured WEHI-3B D+ murine myelomonocytic and HL-60 human promyelocytic leukemia cells with millimolar concentrations of lithium chloride resulted in concentration-dependent increases in the number of differentiated myeloid cells, as determined by the ability of the cells to reduce nitroblue tetrazolium and by the binding of myeloid specific antibodies, and was associated with an inhibition of cellular proliferation. The effects of lithium chloride on growth and differentiation were antagonized by KCl, whereas NaCl had little effect. The induction of leukemic cell maturation by lithium chloride was markedly enhanced by the addition of low levels of retinoic acid. In contrast, other differentiation inducing agents (i.e. dimethyl sulfoxide and selenazofurin) had no effect on the degree of maturation induced by lithium. These findings suggest that the combination of lithium chloride and retinoic acid may have clinical utility in the treatment of leukemia through the induction of terminal differentiation.

Human myelomonocytic leukemic cell line, designated as KY-821, and its sublines KY-Ra, KY-VCR, and KY-MTX, which were resistant to cytosine arabinoside, vincristine, and methotrexate, respectively, were compared for response to various hematopoietic growth factors. Cells of KY-Ra and KY-VCR proliferated in response to natural interleukin-1 (nIL-1), whereas the proliferation of KY-821 and KY-MTX was inhibited. Unexpectedly, recombinant IL-1 alpha and IL-1 beta had no effect on the proliferation of each cell line. The effect of nIL-1 was partially deleted by an addition of optimal anti-IL-1. Supernatants of each cell line had no IL-1 activity. Interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha) also had an inhibitory effect for KY-821 and KY-MTX, but lacked such effect in KY-RA and KY-VCR. nIL-1, IFN gamma and TNF alpha could not differentiate between any of the cell lines but IFN gamma and TNF alpha induced monocytic surface antigens. In addition, there was no difference in the number of IL-1 and TNF alpha receptors in each cell line. These results indicate that there is a difference in biological effects between nIL-1 and recombinant IL-1 species and acquirement of resistance for some types of drugs may associate closely with different responses to hematopoietic growth factors, probably through altered postmembranous transduction.

Retinoic acid (RA) has been shown to be an inducer of the terminal differentiation of several leukemia cell lines in vitro and in clinical trials to produce a high percentage of remissions in patients with acute promyelocytic leukemia. In an effort to increase the therapeutic efficacy of RA, we have measured the capacity of granulocyte colony-stimulating factor (G-CSF) to enhance the differentiation inducing activity of RA in WEHI-3B D+ monomyelocytic leukemia cells. Combinations of G-CSF and RA produced a supra-additive increase in the percentage of WEHI-3B D+ cells reducing nitro blue tetrazolium and expressing Mac-1 (CD11b) antigen on the cell surface, two markers of the mature state. In the presence of 50 ng/ml of G-CSF, which produced only 12% differentiation when used alone, 0.5 microM RA induced the same degree of cellular differentiation as the optimum concentration of RA (i.e. 7 microM) employed alone. The supra-additive differentiation produced by this combination was prevented by the presence of G-CSF monoclonal antibody in the culture medium, resulting in a degree of maturation comparable to that produced by the retinoid alone. When cells were sequentially exposed to G-CSF followed by RA, a much higher level of differentiation was obtained than when the order was reversed, suggesting that WEHI-3B D+ cells were primed to enter a differentiation pathway by G-CSF. The supra-additive terminal differentiation exhibited by the mixture of G-CSF and RA suggests that these agents should be evaluated for the therapeutic efficacy of the combination in patients with acute non-lymphocytic leukemia.

The chemicals 6-mercaptopurine (6-MP) and methotrexate (MTX) are the cornerstones in the maintenance treatment of acute lymphoblastic leukemia. The intracellular metabolism of 6-MP to 6-thioguanosine nucleotides (TGN) via 6-thioinosine 5'-monophosphate (TIMP) is crucial for its cytotoxic effect. MTX inhibits purine de novo synthesis and thereby increases the intracellular PRPP being a substrate for the phosphoribosylation of 6-MP to TIMP. Hypoxanthine has been shown to inhibit the uptake of 6-MP over the cell membrane and the phosphoribosylation of 6-MP to TIMP. We have previously shown that the conversion of TIMP to TGN decreases at 6-MP concentrations above 5 microM in vitro. The aim of the present study was therefore to investigate the effect of MTX increasing the PRPP and TIMP concentrations and of hypoxanthine decreasing the TIMP concentration on the formation of TGN from TIMP. Murine myelomonocytic leukemia cells (WEHI-3b) were treated with 6-MP in vitro. The drug concentration was kept constant by continuous addition of 6-MP during the experiment. With this technique, the concentration of TGN begins to decrease already at 6-MP concentrations above 2 microM. The addition of 0.2 microM MTX 6 h before 6-MP strongly inhibited the purine de novo synthesis, decreased the ATP, and increased the PRPP concentration 4-fold. The intracellular concentrations of TIMP and to a lesser extent TXMP also increased. The concentrations of the TGN were, however, basically unaffected by the preincubation with MTX. Simultaneous addition of 20-50 microM hypoxanthine and 6-MP decreased the accumulation of all cellular 6-MP metabolites. It is concluded that the synergistic cytotoxic effect of the combination of 6-MP and MTX is not based on biochemical modulation of the 6-MP metabolism by MTX.

The activities of protein tyrosine kinase and phosphatidylinositol turnover have been found to be associated with cell growth and differentiation. We examined the effects of some inhibitors for these biochemical activities in human myelogenous leukemia cells. Genistein, which is known to inhibit the activities of protein tyrosine kinase, phosphatidylinositol turnover and topoisomerase II, induced nitroblue tetrazolium (NBT) reduction and lysozyme activity in ML-1, HL-60 and U937 cells. Morphological studies showed that genistein-induced differentiation of myeloblastic ML-1 cells into promyelocytes and of promyelocytic HL-60 cells into mature granulocytes. The differentiation-inducing effect of genistein was augmented by addition of 1 alpha,25-dihydroxyvitamin D3 (VD3) or retinoic acid, VD3 being more effective than retinoic acid. Methyl 2,5-dihydroxycinamate, a protein tyrosine kinase inhibitor, had only a weak effect in inducing differentiation of ML-1 cells. On the other hand, psi-tectorigenin was more effective than genistein in inducing the differentiations of ML-1 and HL-60 cells. Psi-tectorigenin is reported to inhibit phosphatidylinositol turnover without inhibiting protein tyrosine kinase. Thus modulation of phosphatidylinositol turnover might be more important than that of protein tyrosine kinase activity for differentiation of some myelogenous leukemia cells.

Freshly obtained leukemic cells from 64 patients with acute myeloid leukemia were investigated. Intracellular protein and enzyme content (myeloperoxidase (MPO), acid esterase with alpha naphthyl acetate (ANAE) and alpha naphthyl butyrate (ANBE) and lactate dehydrogenase) were measured together with the cell diameter and compared with FAB-classification, cytochemical staining and clinical features. Mean values of MPO and acid esterase discriminate between the pure myeloid leukemias, although overlap between various subgroups is considerable. No correlation between cytochemical and quantitative enzyme determination was found. The logistic regression analysis revealed a dependency of the occurrence of complete remission on protein and ANBE (p less than 0.01) content. We concluded that quantitative enzyme determination is an easily measurable parameter of maturation and may have prognostic significance.

The sera of 28 patients with acute myeloid leukemia AML--subtypes M1 to M6 were screened for human prolactin (h-PRL). Serum TSH (thyroid stimulating hormone), LH (luteinizing hormone), FSH (follicle stimulating hormone), beta-estradiol, free T4 (tetra-iodotyronine) and testosterone were also determined. It was found that in 16 of the 28 patients h-PRL was significantly elevated while all other endocrine values were normal. In a patient subtype M4 with elevated serum h-PRL we demonstrate by immunoblotting that the hormone and its dimer are present in the blast cells. This may reflect ectopic synthesis due to altered expression of homeobox genes and a requirement of h-PRL as a growth stimulant of the leukemic myeloblast.

Nineteen of 71 (26%) cases of acute myelogenous leukemia (AML) were found to express CD7, a cell surface marker found early during T lineage differentiation. These myeloid leukemias often expressed other lymphoid markers and frequently had rearranged T-cell receptor beta and immunoglobulin heavy chain genes. We propose that in CD7+ AML, the malignant transformation occurred in a CD7+ progenitor cell. CD7+ myeloid leukemic precursors may be capable of limited differentiation with loss of CD7 during the initial phase of the disease, but this capacity may diminish during the course of treatment such that CD7 expression persists.

The role of hematopoietic growth factors in the pathogenesis of human leukemias is still obscure. In this study, RNA from 24 human acute myelomonocytic leukemias (AML) was used to analyze the expression of the macrophage colony stimulating factor (M-CSF) and its corresponding receptor (c-fms). Fifty percent of AML cells exhibited c-fms transcripts of regular length but at a lower level than in normal monocytes/macrophages. In most cases the reduced c-fms expression of AML cells was not associated with autostimulatory M-CSF expression. Only a few cases of AML showed co-expression of M-CSF and c-fms, which by contrast was regularly observed in cultivated blood monocytes and some tissue macrophage subsets. Higher levels of c-fms expression could be found in AMLs with a more mature monocytic immunophenotype. Permanent myelomonocytic cell lines expressed c-fms only after induction of monocytic differentiation. Neither the M-CSF gene nor the c-fms gene were rearranged in AML cells. In AML cells the homozygote genotype of the c-fms gene predominated. Our results do not provide evidence for the involvement of M-CSF and c-fms genes in human myeloid leukemogenesis. c-fms expression appears to indicate monocytic differentiation within the myelomonocytic lineage. We found autostimulatory M-CSF expression to be a physiologic feature of some tissue macrophages and hence not necessarily associated with neoplastic proliferation.

Cells of the human myelomonocytic line RC-2A can be induced to differentiate towards mature monocytes by culture in the presence of phytohaemagglutinin-treated lymphocyte conditioned medium (Lyons and Ashman, Leukemia Res. 11, 797, 1987). We have now examined the effect on RC-2A cells of some (recombinant) cytokines which might be present in conditioned medium. Gamma interferon most closely mimicked the effect of conditioned medium in inducing clonogenic suppression and the induction of monocytic maturation over 7 days of culture. Granulocyte colony stimulating factor induced enhancement of proliferation followed by clonogenic suppression, while granulocyte-macrophage colony stimulating factor had a purely stimulatory effect on proliferation over a 7-day period. Tumour necrosis factor alpha failed to affect cell proliferation or to induce characteristic monocytic differentiation, but did increase the expression of C3bi receptors. We conclude that RC-2A cells have receptors for all four cytokines studied, and that gamma interferon is a major differentiation-inducing stimulus for these cells.

We investigated the relationship between the expression of surface antigens and the cell cycle phase in leukemic cells from cell lines and one patient using two-color flow cytometry, in order to determine the reason for the uneven expression of some markers which frequently leads to equivocal results as to leukemic phenotyping. As a result, it was demonstrated that monocyte-related differentiation markers, including I2, My4, Mo1 and Mo2, on monocytoid leukemic cells are preferentially expressed at the G0/G1 phase. Consequently, it is expected that the positivities for such markers vary with the proliferation status of the leukemic cells.