G-CSF signaling can differentiate promyelocytes expressing a defective retinoic acid receptor: evidence for divergent pathways regulating neutrophil differentiation

Several lines of investigation suggest that granulocyte colony-stimulating factor (G-CSF) augments all-trans retinoic acid (ATRA)-induced neutrophil differentiation in acute promyelocytic leukemia (APL). We sought to characterize the relationship between G-CSF- and ATRA-mediated neutrophil differentiation. We established a G-CSF receptor-transduced promyelocytic cell line, EPRO-Gr, derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent EPRO cell line harboring a dominant-negative retinoic acid receptor alpha (RARalpha). In EPRO-Gr, neutrophil differentiation occurs either in GM-CSF upon addition of ATRA or upon induction with G-CSF alone. Transient transfection of EPRO-Gr cells with a RARE-containing reporter plasmid demonstrates increased activity in the presence of ATRA, but not G-CSF, while STAT3 phosphorylation occurs only in response to G-CSF. This suggests that ATRA-mediated differentiation of EPRO-Gr cells occurs via a RARE-dependent, STAT3-independent pathway, while G-CSF-mediated differentiation occurs via a RARE-independent, STAT3-dependent pathway. ATRA and G-CSF thus regulate differentiation by divergent pathways. We characterized these pathways in the APL cell line, NB4. ATRA induction of NB4 cells resulted in morphologic differentiation and up-regulation of C/EBPepsilon and G-CSFR, but not in STAT3 phosphorylation. The addition of G-CSF with ATRA during NB4 induction resulted in STAT3 phosphorylation but did not enhance differentiation. These results may elucidate how G-CSF and ATRA affect the differentiation of primary and ATRA-resistant APL cells.     

Retinoids and myelomonocytic growth factors cooperatively activate RARA and induce human myeloid leukemia cell differentiation via MAP kinase pathways

Use of all-trans-retinoic acid (ATRA) in combinatorial differentiation therapy of acute promyelocytic leukemia (APL) results in exceptional cure rates. However, potent cell differentiation effects of ATRA are so far largely restricted to this disease and long-term survival rates in non-APL acute myelogeneous leukemia (AML) remain unacceptably poor, requiring development of novel therapeutic strategies. We demonstrate here that myelomonocytic growth factors (granulocyte colony-stimulating factor [G-CSF] and/or granulocyte macrophage colony-stimulating factor [GM-CSF]) potentiate differentiation effects of ATRA in different AML cell lines and primary cells from patients with myeloid leukemia. The ligand-dependent activities of endogenous and transiently expressed retinoic acid receptor alpha (RARalpha) isoforms can be potentiated by G/GM-CSF in U-937 cells and correlate with increased expression of ATRA-inducible RARalpha2 isoform. Specific inhibitors of mitogen mitogen-activated protein kinase (MAPK) (MEK)-1/-2 or p38 extracellular signal-related kinase (ERK) kinase diminish the ATRA as well as ATRA and G/GM-CSF-induced activation of the RARalpha proteins and decreased the differentiation-induced decline in cell numbers. Our data demonstrate that acting, at least in part, via the MAP kinase pathways, myelomonocytic growth factors enhance ATRA-dependent activation of the RARalpha isoforms and maturation of myeloid leukemia cells. These results suggest that combinatorial use of these agents may be effective in differentiation therapy of AML.     

In vitro effect of granulocyte-colony stimulating factor and all-trans retinoic acid on the expression of inflammatory cytokines and adhesion molecules in acute promyelocytic leukemic cells.

Differentiation therapy with all-trans retinoic acid (ATRA) represents a landmark approach in the treatment of acute promyelocytic leukemia (APL). However, a potentially fatal complication of retinoic acid (RA) syndrome occurs in about a quarter of patients and its pathophysiology is still unclear. In order to investigate whether or not the treatment with ATRA leads to increased elaboration of inflammatory cytokines and adhesion molecules by the APL cells, the expression of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-8, L-selectin and intercellular adhesion molecule-1 (ICAM-1) was examined in the APL cells after induction of differentiation with ATRA in the presence or absence of granulocyte-colony stimulating factor (G-CSF) or IL-3 in the present study. Cytokine elaboration by the treated cells was detected using both Northern blotting and enzyme-linked immunosorbent assay. Our results have shown that ATRA induces an increased expression of IL-8, IL-1beta, TNF-alpha and ICAM-1 in APL cells, which can be amplified by the addition of G-CSF. These data imply that the induction of inflammatory cytokines in APL cells may play an important role in the pathogenesis of RA syndrome. Furthermore, G-CSF, through its potent differentiating activity, may increase the risk of such complications during ATRA treatment.     

Histone deacetylase inhibitor but not arsenic trioxide differentiates acute promyelocytic leukaemia cells with t(11;17) in combination with all-trans retinoic acid

Acute promyelocytic leukaemia (APL) with t(11;17)/PLZF-RARalpha responds poorly to all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3), in contrast to APL with t(15;17)/PML-RARalpha. Molecular studies have shown that histone deacetylase (HDAC) recruited by PLZF-RARalpha is associated with the ATRA resistance. Here, we analysed in vitro the differentiation of APL cells with t(11;17) using ATRA, As203, granulocyte colony-stimulating factor (G-CSF), HDAC inhibitor trichostatin A (TSA), or combinations of these. Although 1 microM ATRA, which stimulated the differentiation of APL cells with t(15;17), was insufficient to induce differentiation, 3 microM ATRA induced terminal differentiation into granulocytes. As203 alone or in combination with ATRA induced neither differentiation nor apoptosis. However, the combination of TSA and 1 microM ATRA had a potent differentiating effect, although TSA alone had little effect. The combination of 1 microM ATRA and G-CSF did not induce differentiation. These results indicate that APL cells with t(11;17) need a higher concentration of ATRA than those with t(15;17) to differentiate and suggest that HDAC inhibitor is a promising differentiation enhancer in APL with t(11;17).     

Acute myelogenous leukemia with a t(2;17;4)(p13;q21;p16) aberration: effective treatment with all-trans retinoic acid and granulocyte colony-stimulating factor

The efficacy of all-trans retinoic acid (ATRA) in patients with acute promyelocytic leukemia (APL) has been well documented. However, ATRA is not as effective against other types of acute myelogenous leukemia (AML) or myelodysplastic syndromes. We present a patient with AML (FAB: M2) associated with a t(2;17;4)(p13;q21;p16) chromosomal defect in which the 17q21 breakpoint was not within the retinoic acid receptor alpha locus which is typically rearranged in APL. This patient was successfully treated with ATRA and granulocyte colony-stimulating factor and improvement of hematological parameters lasted for 19 months without the use of cytotoxic agents.     

Human granulocyte-monocyte colony-stimulating factor and interleukin 3 stimulate monocyte cytotoxicity through a tumor necrosis factor- dependent mechanism

Human colony-stimulating factors (CSF) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells. In this study, the effects of three recombinant human CSFs (granulocyte- monocyte CSF [GM-CSF], interleukin 3 [IL-3], and granulocyte CSF [G- CSF]) on antibody-independent monocyte tumoricidal activity were investigated by using WEHI 164 fibrosarcoma cells as monocyte-sensitive targets. None of the CSFs directly induced monocyte cytotoxicity, although both GM-CSF and IL-3 were found to significantly enhance monocyte killing in response to a second stimulatory event (endotoxin). No effect was seen with G-CSF. Antitumor necrosis factor antibody completely abolished CSF-enhanced monocyte cytotoxicity, which suggests that this effect was mediated through increased release of tumor necrosis factor (TNF). As previously shown for GM-CSF, IL-3 was found to induce cytoplasmic accumulation of TNF messenger RNA (mRNA) after 18 hours of exposure. These results suggest that GM-CSF and IL-3 may stimulate monocyte killing indirectly by enhancing expression of TNF mRNA, thereby leading to augmented TNF protein secretion in response to a second activation signal.     

Retinoids--"differentiation agents" for cancer treatment and prevention.

The ability of vitamin A and its derivatives to induce differentiation in certain target tissues has been appreciated for nearly a century. Recently, oral all-trans retinoic acid (ATRA), a vitamin A metabolite, has been shown to induce terminal differentiation of leukemic cells in patients with acute promyelocytic leukemia (APL). Complete remissions are obtained and normal hematopoiesis is established in an outpatient setting with minimal side effects in the majority of cases. Although remissions are not durable, disseminated intravascular coagulation, a frequent complication of remission induction in APL, is avoided by oral ATRA prior to definitive chemotherapy. The molecular basis for the efficacy of ATRA in APL appears to be the involvement of the retinoic acid receptor alpha locus in the t(15;17) translocation breakpoint characteristic of APL.     

AM580, a stable benzoic derivative of retinoic acid, has powerful and selective cyto-differentiating effects on acute promyelocytic leukemia cells

All-trans retinoic acid (ATRA) is successfully used in the cyto- differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically, APL cells express PML-RAR, an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia- specific t(15;17) chromosomal translocation. We show here that AM580, a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist, is a powerful inducer of granulocytic maturation in NB4, an APL-derived cell line, and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF), the compound induces granulocytic maturation, as assessed by determination of the levels of leukocyte alkaline phosphatase, CD11b, CD33, and G-CSF receptor mRNA, at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast, AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells, two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580, whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments, using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha, show that AM580 has a lower affinity than ATRA for both receptors. However, in the presence of PML-RAR, the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid, whereas, in the presence of RAR alpha, AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells.     

Interleukin-1 alpha upregulates tumor necrosis factor receptors expressed by a human bone marrow stromal cell strain: implications for cytokine redundancy and synergy

To explore the biochemical and physiologic basis of the overlapping effects of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha) on myeloid cytokine production, we have studied the dynamics of granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) production as well as IL-1 receptor and TNF receptor expression in a clonally derived bone marrow stromal cell strain (CDCL). IL-1 alpha and TNF alpha act in a synergistic manner to stimulate G-CSF and GM-CSF production by CDCL, resulting in an increase in CSF secretion that is 250-fold greater than that observed with either cytokine alone. This synergism in protein secretion is paralleled by synergistic increases the steady-state level of GM- and G-CSF mRNA, with supra-additive levels achieved by 24 hours. Coincident with this synergistic induction of myeloid CSFs, treatment of CDCL cells with IL-1 alpha induces a 300% increase in the expression of TNF receptors. IL-1 alpha induction of TNF receptors reaches a peak after 6 hours and gradually returns to baseline level by 24 hours. IL-1 alpha does not affect TNF receptor ligand binding affinity. A kinetic study comparing IL-1/TNF synergistic induction of growth factor secretion with IL-1 alpha induction of TNF receptors shows that these events occur in parallel. In contrast with the induction of TNF receptors by IL-1 alpha, treatment with TNF alpha has no effect on either the number of IL-1 receptors expressed by CDCL cells or IL-1 receptor ligand binding affinity. Brief treatment of IL-1 alpha/TNF alpha-stimulated CDCL cells with cycloheximide before receptor induction reduces the synergistic increase in growth factor mRNA by 40% to 60% compared with cells not treated with CHX. Taken together, these results raise the possibility that IL-1 alpha cross-induction of TNF receptors may contribute to the biochemical mechanisms underlying the synergistic stimulation of G-CSF and GM-CSF production by IL-1 alpha and TNF alpha.     

Granulocyte colony-stimulating factor and its receptor in acute promyelocytic leukemia

Expression of granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) and in vitro proliferative response to G-CSF were investigated by quantitative immunofluorescence and [³H] thymidine uptake, respectively, in a series of acute myeloid leukemias (AML). The results indicated that G-CSFR was detected at high levels in acute promyelocytic leukemia (APL) cells, in comparison with other types of AML. Moreover, APL cells were also seen to predominantly proliferate in response to G-CSF. Based on these observations, we administered recombinant human G-CSF to a patient with APL in the third relapse that was resistant to both cytotoxic agents and all trans retinoic acid, in an attempt to sensitize the leukemic cells to cell-cycle-dependent agents. Complete remission was achieved. The finding that APL cells are exquisitely responsive to G-CSF supports the view that G-CSF is useful for augmentation of their vulnerability to cell-cycle specific agents. Am. J. Hematol. 58:31–35, 1998. © 1998 Wiley-Liss, Inc.     

The aberrant fusion proteins PML-RAR alpha and PLZF-RAR alpha contribute to the overexpression of cyclin A1 in acute promyelocytic leukemia

Cyclin A1 is a newly discovered cyclin that is overexpressed in certain myeloid leukemias. Previously, the authors found that the frequency of cyclin A1 overexpression is especially high in acute promyelocytic leukemia (APL). In this study, the authors investigated the mechanism of cyclin A1 overexpression in APL cells and showed that the APL-associated aberrant fusion proteins (PML-retinoic acid receptor alpha [PML-RAR alpha] or PLZF-RAR alpha) caused the increased levels of cyclin A1 in these cells. The ectopic expression of either PML-RAR alpha or PLZF-RAR alpha in U937 cells, a non-APL myeloid cell line, led to a dramatic increase of cyclin A1 messenger RNA and protein. This elevation of cyclin A1 was reversed by treatment with all-trans retinoic acid (ATRA) in cells expressing PML-RAR alpha but not PLZF-RAR alpha. ATRA also greatly reduced the high levels of cyclin A1 in the APL cell lines NB4 and UF-1. No effect of ATRA on cyclin A1 levels was found in the ATRA-resistant NB4-R2 cells. Further studies using ligands selective for various retinoic acid receptors suggested that cyclin A1 expression is negatively regulated by activated RAR alpha. Reporter assays showed that PML-RAR alpha led to activation of the cyclin A1 promoter. Addition of ATRA inhibited PML-RAR alpha-induced cyclin A1 promoter activity. Taken together, our data suggest that PML-RAR alpha and PLZF-RAR alpha cause the high-level expression of cyclin A1 seen in acute promyelocytic leukemia. (Blood. 2000;96:3894-3899)     

A unique case of acute promyelocytic leukemia showing monocytic differentiation after ATRA (all-trans retinoic acid) therapy

Acute promyelocytic leukemia (PML) is characterized by a reciprocal translocation between chromosomes 15 and 17 resulting in a chimeric PML and retinoic acid receptor alpha (RARA) oncogene. The resultant fusion protein (PML/RARA) is thought to block differentiation of bone marrow cells arrested at the promyelocytic stage. In vitro and in vivo studies have shown that the large majority of APL cells undergo granulocytic maturation after ATRA therapy. We report a unique case of a PML/RARA positive APL patient exhibiting extensive monocytic differentiation after ATRA therapy as documented by morphology, flow cytometry, and FISH studies. We discuss potential dual capability for granulocytic/monocytic differentiation of PML/RARA positive APL cells and implications of monocytic differentiation in the management of APL patients treated with ATRA.     

Acute promyelocytic leukemia with a PLZF-RARalpha fusion protein

In most cases of acute promyelocytic leukemia (APL), a fusion of the promyelocytic leukemia (PML) and the retinoic acid receptor-alpha (RARalpha) genes occurs, resulting in the expression of a PML-RARalpha chimeric protein. In approximately 1% of the cases of APL, variant chromosomal aberrations may be found fusing RARa with other genes. Four variant mutations have been described, and the t(11;17)(q21;q23) translocation generating a promyelocyte leukemia zinc finger (PLZF)-RARalpha fusion gene is the most common. PLZF-RARalpha-positive APL forms a clinically distinct group because unlike PML-RARalpha-positive leukemia, it does not respond to retinoic acid with terminal granulocytic differentiation of the cells, and remissions cannot be achieved with retinoids alone. At the molecular level, this has been explained by the retinoic acid-insensitive binding of corepressor proteins to the PLZF part of the fusion protein, leading to sustained repression of target genes that are important for cellular differentiation. Targeting of the PLZF-RARalpha-bound corepressor complexes using a combination of all-trans retinoic acid (ATRA) and deacetylase inhibitors has shown that the repression of target genes can be relieved, allowing differentiation of the cells. In addition, when a combination of retinoic acid and the hematopoietic growth factor granulocyte colony-stimulating factor (G-CSF) is applied, the cells may be forced to undergo terminal differentiation, both in vitro and in vivo. This suggests that signals from the activated G-CSF receptor may induce the release of corepressor proteins from PLZF. Together, these findings indicate that PLZF-RARalpha-positive leukemia is not completely resistant to differentiation induction if the proper costimuli are given.     

Induction therapy of acute promyelocytic leukemia with all-trans retinoic acid: a case, followed by conventional post-remission chemotherapy in complete remission]

A 41-year-old man with untreated acute promyelocytic leukemia (APL) was treated with all-trans retinoic acid (ATRA) 80mg/body/day per os. Complete remission was reached in 16 day without bone marrow hypoplasia and aggravated disseminated intravascular coagulation. The chromosomal abnormality, t (15;17), which presented before therapy has not been found since the 29th day of therapy. During the course of induction therapy with ATRA, there was no complication worth of mentioning. Induction therapy with ATRA is thought to be more effective and safer than conventional chemotherapy to attain complete remission in APL. The complete remission has been maintained for 11 months with conventional postremission chemotherapy.     

Differentiation and functional activity of human eosinophilic cells from an eosinophil HL-60 subline: response to recombinant hematopoietic growth factors.

We studied the effect of hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor [GM-CSF], granulocyte [G]-CSF, interleukin (IL)-1, IL-3, IL-5, IL-6, and macrophage [M]-CSF) on differentiation and functional activity of human eosinophilic HL-60 cells (Eos-HL-60) and compared them with effects on parental HL-60 promyelocytic leukemia cells. Purified biosynthetic GM-CSF and IL-5 enhanced cell proliferation and induced eosinophilic differentiation in the eosinophilic subline in both liquid and agar cultures. IL-3 and IL-6 stimulated cell proliferation but had no effect on cell differentiation, whereas IL-1 and G-CSF affected neither differentiation nor proliferation of Eos-HL-60 cells under the conditions tested. GM-CSF-, IL-3-, and IL-5-treated Eos-HL-60 cells showed increased O2- production in response to phorbol esters (PMA), enhanced phagocytosis of Candida albicans, and release of the enzymes arylsulfatase, beta-glucuronidase and eosinophil peroxidase (EPO). The degranulation of eosinophils induced by GM-CSF, IL-5, and IL-3 may have relevance to the potential clinical toxicity of these hematopoietins, which also stimulate eosinophilopoiesis. G-CSF had no effect on enzyme release, oxidative metabolism, or phagocytic capacity of Eos-HL-60 cells. IL-5 did not affect proliferation, differentiation, or enzyme release in promyelocytic HL-60 cells. These results indicate the specificity of IL-5 for the eosinophil lineage, confirm the effects of GM-CSF and IL-3 on eosinophilopoiesis and mature eosinophil function in a model system, and indicate the absence of G-CSF and IL-1 stimulation of eosinophils. The Eos-HL-60 line is a useful model for studying human eosinophil responses to cytokines.     

Treatment of acute promyelocytic Leukemia and other hematologic malignancies with arsenic trioxide: Review of clinical and basic studies

Acute promyelocytic leukemia (APL) is now the most potentially curable subtype of acute myeloid leukemia in adults because of the introduction of novel approaches in the management of this disease. All-trans-retinoic acid (ATRA)-based therapy is now the first-choice treatment of patients presenting with de novo APL, and clinical studies have shown that nearly all patients who receive ATRA therapy achieve complete remission. However, approximately 20% to 30% of APL patients eventually have relapses with resistance to further ATRA treatment. Arsenic trioxide (As2O3 [ATO]) has been established as highly effective therapy for patients with APL, even for those with disease refractory to ATRA. Furthermore, results of recent studies have suggested a broad therapeutic potential for ATO in the treatment of hematologic malignancies beyond APL. In this review, we discuss the clinical activity and multiple mechanisms of ATO therapy in the management of APL and other hematologic neoplasms.     

Hematopoietic myeloid cell differentiation diminishes nucleotide excision repair

Myeloid cell differentiation is the process by which stem cells develop into mature monocytes or granulocytes. This process is achieved by the sequential activation of variety of genes. Disruption of this process can result in immunodeficiency, bone marrow failure syndrome, or leukemia. Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation and can be treated by a combination of all-trans retinoic acid (ATRA) and anthracycline. This treatment can induce leukemic cell differentiation, leading to extremely high remission rates. XAB2, a molecule involved in nucleotide excision repair (NER), is downregulated during granulocyte differentiation and shows reduced expression in NB4 APL-derived cells in vitro. Differentiation of APL by ATRA treatment reduced XAB2 expression levels in vivo. These observations suggest that cellular differentiation is associated with reduced NER activity and provides new insights into combined differentiation induction. NB4 cells were more susceptible than the immature myeloid leukemic cell lines, Kasumi-3 and Kasumi-1, to the DNA interstrand crosslinking agent cisplatin.     

Thrombocytosis in patients with acute promyelocytic leukaemia during all-trans retinoic acid treatment

In 26 acute promyelocytic leukaemia (APL) patients treated with all- trans retinoic acid (ATRA), 23% had platelet counts between 459 and 800 ×10⁹/l during treatment. These values, observed between days 28 and 45 of ATRA treatment, were transient and asymptomatic. We report two APL cases with platelet counts >1000 × 10⁹/l during ATRA therapy who were treated with recombinant interferon alpha. In both cases ATRA doses were not modified, no complications secondary to thrombocytosis were seen, and they subsequently achieved complete remission. It is suggested that IL-6 may play an important role in the pathogenesis of the thrombocytosis induced by ATRA. To our knowledge, this is the first report of thrombocytosis occurring during ATRA treatment.     

Complete remission through blast cell differentiation in PLZF/RARalpha-positive acute promyelocytic leukemia: in vitro and in vivo studies

Acute leukemia with the t(11;17) expressing the PLZF-RARalpha gene fusion is a rare variant of acute promyelocytic leukemia (APL) that has been associated with poor clinical response to all-trans retinoic acid (ATRA) treatment. However, some recent reports have put into question the absolute refractoriness of this leukemia to ATRA. We describe here a patient with PLZF/RARalpha APL who was treated at relapse with ATRA and low-dose hydroxyurea. Complete hematologic remission was obtained through differentiation of leukemic blasts, as proven by morphologic, immunophenophenotypic, and genetic studies carried out in sequential bone marrow samples. Moreover, in vitro studies indicated that blast differentiation was potentiated by the addition of the histone deacetylase inhibitor tricostatin A, but not of hydroxyurea, to ATRA. Our findings indicate that the maturation block may be overcome and terminal differentiation obtained in this leukemia subset and support the view that sensitivity/refractoriness of this form to ATRA should be revisited.