Effects of novel uracil analogs on proliferation and differentiation of human myeloid leukemia cells.

Twenty-seven novel nucleobases and nucleosides were synthesized by structural modification of uracil, and their effects on growth and differentiation of human myeloid leukemia HL-60 cells were examined. Some of the compounds inhibited the growth of HL-60 effectively. The nitroblue tetrazolium (NBT)-reducing activities of cells treated with the concentrations of these compounds for 50% inhibition of growth were compared. TI-66 (2,4-dibenzyl-6-fluoro-7,7,8,8-tetramethyl-cis-2,4-diazabicyclo-[4.2.0] octane-3,5-dione) was the most effective inducer of NBT-reducing activity and morphological differentiation of HL-60 cells into cells of the myelomonocytic lineage. TI-66 was also effective for induction of differentiation of another human myelogenous leukemia cell line, ML-1 cells, but not for differentiation of human erythroid leukemia K562 or HEL cells, or monocytic U937 cells. The effect of TI-66 in inducing differentiation of HL-60 cells was additive or more than additive in combination with retinoic acid or vitamin D3. Adenine or hypoxanthine alone induced NBT-reducing activity of the cells, and at suboptimal concentrations these compounds enhanced the effect of TI-66, but the enhanced NBT-reducing activities did not exceed the maximal activity induced by TI-66 alone. Simultaneous treatment of HL-60 cells with hypoxanthine reduced the growth inhibition by TI-66 alone. TI-66 was about 150 times more potent on a molar basis than adenine in inducing differentiation of HL-60 cells. These results suggest that nucleobase analogs such as TI-66 should be useful for differentiation therapy of some types of myelogenous leukemia.     

Retinoid X receptor-selective ligands produce malformations in Xenopus embryos.

Retinoids exert pleiotropic effects on the development of vertebrates through the action of retinoic acid receptors (RAR) and retinoid X receptors (RXR). We have investigated the effect of synthetic retinoids selective for RXR and RAR on the development of Xenopus and zebrafish embryos. In Xenopus, both ligands selective for RAR and RXR caused striking malformations along the anterior-posterior axis, whereas in zebrafish only ligands specific for RAR caused embryonic malformations. In Xenopus, RAR- and RXR-selective ligands regulated the expression of the Xlim-1, gsc, and HoxA1 genes similarly as all-trans-retinoic acid. Nevertheless, RXR-selective ligands activated only an RXR responsive reporter but not an RAR responsive reporter introduced by microinjection into the Xenopus embryo, consistent with our failure to detect conversion of an RXR-selective ligand to different derivatives in the embryo. These results suggest that Xenopus embryos possess a unique response pathway in which liganded RXR can control gene expression. Our observations further illustrate the divergence in retinoid responsiveness between different vertebrate species.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro. II. Acute myelogenous leukemia

Immature myeloid cells prepared from patients with AML were placed into suspension culture and studied over a 2-week period. Cell numbers usually fell and viability was not well maintained. Some degree of differentiation was observed in most cultures. Considering these observations, together with those derived from parallel studies of immature CML cells, the data suggest that AML cells are more dependent than CML cells on environmental conditions for the maintenance of cell viability and proliferation. This is especially the case for AML cells obtained at the time of initial diagnosis. On the other hand, AML cells and myeloid blastic crisis CML cells are similar with respect to their apparent greater ability to differentiate in vitro than in vivo. The addition of recombinant hemopoietins to the suspension cultures of AML cells is associated with either increased proliferation or differentiation but not both. The cells of different patients respond differently to the different hemopoietins and the different hemopoietins produce different affects in cultures of cells obtained from the same patient.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro. I. Chronic myelogenous leukemia

Immature cells prepared from chronic myelogenous leukemic patients in the chronic or blastic phase of their disease were placed into suspension culture in vitro and studied over a 2-week period. Regardless of the status of the disease, the immature cells proliferated and maintained good viability. In contrast, evolution of the disease toward blastic crisis was associated with a progressive loss in the ability of the immature cells to differentiate. Cell proliferation and differentiation were independent of added growth factors.     

Pulsatilla saponin A induces differentiation in acute myeloid leukemia in vitro

Objectives: To identify whether Pulsatilla saponin A (PsA), an active molecule extracted from Pulsatilla chinensis regel, can induce acute myeloid leukemia (AML) cells differentiate.

Methods: PsA was isolated from P. chinensis, and its effects of differentiation induction on both AML cell lines and the primary leukemia cells were investigated.

Results: Compared with the untreated control, PsA induced the differentiation of U937 cells, K562 cells and HL-60 cells, represented as the increased CD15+ cells in a dose- and time-dependent manner in all the three AML cell lines, after PsA treatment. As the same time, the cell morphology of these AML cells was changed correspondingly; the cytoplasm/nuclei ratio was increased, basophilic cytoplasm was decreased, and eccentric nucleus and granules were also observed. Also, the same effects of differentiation induction by PSA were confirmed in the primary leukemia cells. However, the specific MEK/ERK inhibitor U0126 effectively abrogated the differentiation induced by PsA in vitro.

Conclusions: PsA can modify the differentiation activity of AML cells, probably though the MEK/ERK signaling pathway.     

Gefitinib induces myeloid differentiation of acute myeloid leukemia

Cure rates for patients with acute myeloid leukemia (AML) remain low despite ever-increasing dose intensity of cytotoxic therapy. In an effort to identify novel approaches to AML therapy, we recently reported a new method of chemical screening based on the modulation of a gene expression signature of interest. We applied this approach to the discovery of AML-differentiation-promoting compounds. Among the compounds inducing neutrophilic differentiation was DAPH1 (4,5-dianilinophthalimide), previously reported to inhibit epidermal growth factor receptor (EGFR) kinase activity. Here we report that the Food and Drug Administration (FDA)-approved EGFR inhibitor gefitinib similarly promotes the differentiation of AML cell lines and primary patient-derived AML blasts in vitro. Gefitinib induced differentiation based on morphologic assessment, nitro-blue tetrazolium reduction, cell-surface markers, genome-wide patterns of gene expression, and inhibition of proliferation at clinically achievable doses. Importantly, EGFR expression was not detected in AML cells, indicating that gefitinib functions through a previously unrecognized EGFR-independent mechanism. These studies indicate that clinical trials testing the efficacy of gefitinib in patients with AML are warranted.     

Effects of various recombinant human hematopoietic growth factors on proliferation of blast cells in acute myeloid leukemia in vitro]

In vitro proliferative response of the blast cells from 21 AML patients to hematopoietic growth factors (IL-3, GM-CSF, G-CSF and MCSF) was investigated. Proliferation of AML cells in the majority of cases was induced or promoted by one or more CSFs, among which the stimulation of IL-3 was the most effective. Spontaneous proliferation of the blast cells was also observed in half of the cases and could be inhibited as well as promoted by some CSFs. It is suggested that in vitro proliferation of AML cells varies from patient to patient and that CSF plays important roles in leukemogenesis.     

Stimulation of proliferation and differentiation of acute myeloid leukemia cells on a bone marrow stroma in culture

Purified acute myeloid leukemia (AML) blasts were placed in Dexter cultures with and without a preestablished normal bone marrow stroma to investigate whether their proliferation was dependent on a stroma, as is the case with normal marrow cells. It was also studied whether AML cells were induced to differentiate under a stromal influence. Ten patients with untreated AML were included in the study. In most cases, AML cells were maintained at higher levels when a normal stroma was present. Cell recoveries varied widely between patients. Net cell production was apparent in nine of ten stroma cultures. In most paired cultures without a preestablished stroma, cell numbers decreased. Leukemic colony-forming cells (L-CFU) (clonogenic cells) were also recovered in higher numbers from stroma cultures, but decreased rapidly. The peak of L-CFU preceded that of AML nucleated cells. Cytogenetic analysis (three cases) confirmed the AML karyotype of cultured cells. AML blasts were tested weekly with a number of monoclonal antibodies. During culture, the percentage of cells expressing mature monocytic and granulocytic surface antigens increased markedly, suggesting progressive maturation. Morphologic maturation at the same time was incomplete. Thus, AML cells proliferate and differentiate in vitro under the influence of a normal bone marrow stroma, a property which these cells share with normal bone marrow cells.     

Differentiation induction of myeloid leukemia cells by glucocorticoid--the differentiation induction effect in vitro and in vivo

The effects of glucocorticoid on the differentiation of myeloid leukemia cells were examined. Dexamethasone at 10(-6)M or 10(-7)M revealed marked effects not only on leukemic blasts' survival but also on its' differentiation in vitro. In 10 of 17 cases of myeloid leukemia, the obvious morphological and functional differentiation induction effects were observed in vitro. The direction of differentiation were differed in leukemia cell lineage and in cases. Granulocytic differentiation in M2 cells, mono-macrophage differentiation in M5 cells and either granulocytic or monocytic differentiation in M4 cells were induced. A AML (M2), it's leukemia cells were induced into granulocytic pathway by dexamethasone in vitro, was treated with prednisolone (40 mg per day).Ara-C (15 mg per day). The increase in peripheral leukocyte count and the decrease in immature cells were observed simultaneously. The peripheral leukocytes mainly consisted of intermediate forms of granulocytes and Pelger-Hu?t like neutrophils probably originated from leukemia cells. After that course, abnormal clone was eliminated from bone marrow. A AMoL, it's leukemia cells were induced into macrophage like cells completely by dexamethasone in vitro, was treated with prednisolone (30 mg per day) and complete remission was obtained without passing through a hematological nadir. It is indicated that anti-tumor effects of glucocorticoid on myeloid leukemia cells are closely related to it's differentiation inducing effect and glucocorticoid can be used as the drug intending for the differentiation induction therapy of acute myeloid leukemia.     

盐酸肾上腺素对白血病细胞株K562增殖及分化的影响

目的探讨盐酸肾上腺素在白血病发生、发展中的作用。方法采用MTT法集落培养实验观察盐酸肾上腺素对白血病细胞株K562细胞增殖的影响;采用Gimesa染色观察细胞形态学变化,采用流式细胞术检测CD71分化抗原表达。结果盐酸肾上腺素可明显降低K562细胞增殖、集落生长,并呈浓度依赖性;在形态上可诱导细胞趋向红系分化,同时上调K562细胞膜CD71分化抗原的表达。结论盐酸肾上腺素在白血病发生、发展中起重要作用,其机制为抑制K562细胞增殖和诱导其分化。     

Induction of differentiation of human myeloid leukemia cells by novel synthetic neurotrophic pyrimidine derivatives

OBJECTIVE: Some pyrimidine analogues have been found to induce differentiation of several human myeloid leukemia cells. Newly synthesized heterocyclic pyrimidine derivatives promote neurite outgrowth and survival in neuronal cell lines. In this study, the growth-inhibiting and differentiation-inducing effects of these pyrimidine derivatives on human myeloid leukemia cells were examined. MATERIALS AND METHODS: Several myeloid leukemia cells were cultured with novel heterocyclic pyrimidine derivatives. Cell differentiation was determined by nitroblue tetrazolium-reducing activity, morphologic changes, expression of CD11b, lysozyme activity, and hemoglobin production. RESULTS: MS-430 (2-piperidino-5,6-dihydro-7-methyl-6-oxo (7H) pyrrolo [2,3-d] pyrimidine maleate) effectively induced HL-60 cells into mature granulocytes. MS-430 activated the mitogen-activated protein kinase (MAPK) of the cells before causing granulocytic differentiation. MAPK activation was necessary for MS-430-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by MS-430. MS-430 also induced monocytic differentiation of THP-1, P39/Tsu, and P31/Fuj leukemia cells, but did not affect erythroid differentiation of K562 or HEL cells. CONCLUSIONS: MS-430 potently induces differentiation of some myelomonocytic leukemia cells. This novel synthesized pyrimidine compound shows promise as a therapeutic agent for treatment of leukemia and as a neurotrophic drug.     

Prognostic implications of basophil differentiation in chronic myeloid leukemia

Basophilia has been reported to indicate an accelerated phase of chronic myeloid leukemia (CML), heralding a poor prognosis. We have studied 47 patients with chronic-phase CML by basophil growth and differentiation assays in vitro, demonstrating an association between basophil growth index (BGI) and clinical time to blast crisis as well as overall survival. In addition to confirming an association between positive BGI and phase of CML in a larger group of patients, a positive BGI predicted death or blast crisis within 2 years' study of chronic phase CML (p less than 0.01), with a sensitivity of 78%, specificity, 81%; the positive predictive value of a positive test, 64%; and a negative predictive value of a negative test, 89%. The survival experience of the 22 evaluable patients with chronic-phase CML and a positive BGI was significantly worse than the survival of the 19 patients with a negative BGI (p less than 0.0001). At 1, 2, and 3 years the proportion of surviving patients with a positive BGI was 0.64, 0.32, and 0.23, respectively, compared with 1.00, 0.90, and 0.79 for those with a negative BGI. The median survival of chronic phase CML patients with a positive test at diagnosis (n = 14) was 27 months versus 54 months for those with a negative diagnosis (n = 14) (p less than 0.05). These findings emphasize the prognostic utility of basophil growth assays in CML and suggest a molecular relationship between leukemic transformation and basophil lineage expression.     

Opposing effects of PI3 kinase pathway activation on human myeloid and erythroid progenitor cell proliferation and differentiation in vitro

OBJECTIVE: To investigate 1) the effects of lineage-specific cytokines (G-CSF and EPO) combined with ligands for different classes of cytokine receptors (common beta chain, gp130, and tyrosine kinase) on proliferation by human myeloid and erythroid progenitor cells; and 2) the signal transduction pathways associated with combinatorial cytokine actions. PATIENTS AND METHODS: CFU-GM and BFU-E were cloned in vitro. Secondary colony formation by replated CFU-GM and subcolony formation by BFU-E provided measures of progenitor cell proliferation. Studies were performed in the presence of cytokine combinations with and without signal transduction inhibitors. RESULTS: Proliferation by CFU-GM and BFU-E was enhanced synergistically when common beta chain receptor cytokines (IL-3 or GM-CSF) were combined with G-CSF or EPO, but not with gp130 receptor cytokines (LIF or IL-6) or tyrosine kinase receptor cytokines (SCF, HGF, Flt-3 ligand, or PDGF). Delayed addition studies with G-CSF+IL-3 and EPO+IL-3 demonstrated that synergy required the presence of both cytokines from the initiation of the culture. The Jak2-specific inhibitor, AG490, abrogated the effect of combining IL-3 with EPO but had no effect on the enhanced CFU-GM proliferation stimulated by IL-3+G-CSF. The PI3 kinase inhibitors LY294002 and wortmannin substituted for G-CSF in combination with IL-3 since proliferation in the presence of LY294002/wortmannin+IL-3 was enhanced to the same extent as in the presence of G-CSF+IL-3. In contrast, LY294002 and wortmannin inhibited proliferation in the presence of EPO and in the presence of EPO+IL-3. CONCLUSION: 1) IL-3 may activate different signal transduction pathways when combined with G-CSF and when combined with EPO; 2) different signal transducing intermediates regulate erythroid and myeloid progenitor cell proliferation; and 3) inhibition of the PI3 kinase pathway suppresses myeloid progenitor cell differentiation and thereby increases proliferation.     

Effects of anti-CD44 monoclonal antibodies on differentiation and apoptosis of human myeloid leukemia cell lines.

Acute myeloid leukemia (AML) is a heterogeneous leukemia characterized by the blockage of myeloid differentiation at different stages, which define distinct AML subtypes. We have recently reported that the ligation of CD44 with 2 activating monoclonal antibodies (mAbs), A3D8 and H90, triggers terminal differentiation of leukemic blasts in AML-M1/2 to AML-M5 subtypes, which are the most frequent ones. However, fresh AML blasts have short in vitro lifespans. Therefore, to find relevant in vitro cellular models for further studying the mechanisms involved in CD44-induced differentiation, we investigated whether CD44 ligation with A3D8 and H90 mAbs can induce terminal differentiation of THP-1, NB4, and HL60 cells, each interesting models of AML-M5 (monoblastic subtype), AML-M3 (promyelocytic subtype), and AML-M2 (myeloblastic subtype), respectively. We also study whether CD44 ligation induces a loss of proliferative capacity, an important feature of late-stage myeloid differentiation. In the second part of our study, we investigated whether A3D8 and H90 anti-CD44 mAbs can induce the differentiation and inhibit the proliferation of KG1a cells, which are very immature AML-M0 blasts. Using functional, antigenic, and cytologic criteria, we presently show that A3D8 and/or H90 induce terminal differentiation of THP-1, HL60, and NB4 cell lines and strongly inhibit their proliferation. Interestingly, cell-specific effects of H90 and A3D8 are observed. We also observe that incubation with A3D8 for 3 to 6 days induces an apoptotic cell death that is moderate in the case of THP-1 and HL60 cells and massive in the case of NB4 cells. Finally, our results demonstrate for the first time that it is possible to reverse the leukemic blockage of KG1a cells by using both an anti-CD44 mAb and retinoic acid. This result may provide a new experimental basis for a differentiative therapy in AML-M0 patients.     

The expression of myeloid-specific antigens on myeloid leukemia cells: correlations with leukemia subclasses and implications for normal myeloid differentiation

The expression of three distinct myeloid-specific cell surface antigens detected by monoclonal antibodies (PMN 6, PMN 29, and AML-2-23) on acute and chronic myeloid leukemia cells is correlated with blast cell morphology and normal myeloid cell antigen display. In studies on normal peripheral blood cells, monoclonal antibodies PMN 6 and PMN 29 have previously been shown to react exclusively with neutrophils while AML-2-23 reacts with both neutrophils and monocytes. The present report demonstrates that these antigens are absent from blast cells of patients with acute myelocytic leukemia (AML) classified as M1 and M2 in the French-American-British system and chronic myelocytic leukemia in myeloid blast crisis. However, leukemia cells with myelomonocytic morphology (M4) expressed all three antigens, while cells with pure monocytic features (M5) were generally only positive for AML-2-23. Based on the absence of these antigens on both leukemic and normal myeloblasts and granulocyte-monocyte progenitors and their characteristic patterns of display on more differentiated leukemic and normal cells, we propose a modified concept of normal myelopoiesis. In this hypothesis, the myeloblast is an uncommitted cell that gives rise to a series of intermediate precursors that acquire committment to either the granulocytic or monocytic lineage marked by the acquisition of specific cell surface markers.