Effects of prostaglandin E2 and all-trans retinoic acid combination on induced differentiation of human acute promyelocytic leukemia NB4 cells

OBJECTIVE: A human promyelocytic leukemia (APL) cell line NB4 was used to demonstrate the synergistic effects between all-trans retinoic acid (ATRA) and prostaglandin E2 (PGE2) on growth inhibition and cytodifferentiation induction. METHODS: NB4 cells were cultured in the presence of either ATRA or PGE2 as a single agent or in combinations at various ratio. Cell growth was measured and myeloid differentiation was tested on consecutive days over the whole course of culture. RESULTS: PGE2 and ATRA synergistically induced the myeloid differentiation of NB4 cells. In comparison with ATRA alone, the combination of PGE2 with ATRA caused an almost 20-fold decrease of effective concentration of ATRA. CONCLUSION: The combination of ATRA and PGE2 at an appropriate ratio may provide a convenient oral regimen of combined differentiation therapy for a better clinical outcome in APL patients.     

All-trans-retinoic acid counteracts endothelial cell procoagulant activity induced by a human promyelocytic leukemia-derived cell line (NB4)

Therapy with all-trans-retinoic acid (ATRA) can rapidly improve the coagulopathy of acute promyelocytic leukemia (APL). This study was designed to evaluate whether the APL cell line NB4 induces the procoagulant activity (PCA) of human endothelial cells (ECs) in vitro, and whether this property is modified after ATRA-induced NB4 maturation. EC monolayers were incubated for 4 hours at 37 degrees C with the conditioned media (CM) of NB4 treated with 1 mumol/L ATRA (ATRA-NB4-CM) or the vehicle (control-NB4-CM). EC lysates were tested for PCA. ATRA-NB4-CM induced significantly more PCA:tissue factor (TF) than control-NB4-CM (P < .01). To identify the cause of TF induction, interleukin (IL)-1 beta antigen levels were measured in CM samples. ATRA-NB4-CM contained significantly more IL-1 beta than control-NB4-CM. EC PCA was significantly inhibited by an anti-IL-1 beta antibody. The addition to the media of 10 mumol/L ATRA counteracted the EC TF expression induced by NB4-CM. These data indicate that ATRA increases the promyelocyte-induced EC TF, partly through increased IL-1 beta production. However, ATRA can protect the endothelium from the procoagulant stimulus of leukemic cells.     

Expression of NADPH oxidase is induced by all-trans retinoic acid but not by phorbol myristate acetate and 1,25 dihydroxyvitamin D3 in the human promyelocytic cell line NB4

Human promyelocytic cells, NB4, differentiate into neutrophils in response to all-trans retinoic acid (ATRA). It has recently been proposed that NB4 cells have bilineage potential because these cells are also able to differentiate into monocyte/macrophages when exposed to a combination of 1,25-dihydroxyvitamin D3 (VD3) and phorbol myristate acetate (PMA). Differentiation of myeloid cells into neutrophils or monocytes is associated with the acquisition of the O2- producing enzyme, NADPH oxidase, which plays a critical role in microbial killing. In this study, the expression of the components of the NADPH oxidase complex during the differentiation of NB4 cells into neutrophils or macrophages has been investigated. Whereas cells exposed to ATRA were able to produce O2- after 2 days of differentiation, they remain unable to generate O2- when exposed to PMA or PMA + VD3. With the exception of p21rac, none of the other oxidase components was expressed in non-differentiated cells. Addition of ATRA induced the progressive expression and accumulation of p22phox, p91phox, p47phox and p67phox. Compared to the other components, p67phox was expressed late and its expression appeared to correlate most closely with the generation of O2- in the differentiation process. In PMA or PMA + VD3-differentiated NB4 cells, expression of the NADPH oxidase components was incomplete. Therefore, ATRA induced the expression of a functional NADPH oxidase complex in neutrophil-like NB4 cells. In contrast, when NB4 cells are exposed to monocytic differentiating agents, they acquire only part of the phenotypic characteristics of monocytes and lack one of the major phagocytic functionalities, the respiratory burst oxidase.     

Relapsed acute promyelocytic leukemia previously treated with all-trans retinoic acid: clinical experience with a new synthetic retinoid, Am-80

All-trans retinoic acid (ATRA), a potent differentiating drug for acute promyelocytic leukemia (APL), induces a high incidence of complete remission (CR) in patients with APL and is now established as a first-line therapy. However, ATRA resistance has become a clinical problem. Patients who relapsed after ATRA-induced CR have had difficulty in obtaining a second CR with ATRA therapy. Although several mechanisms have been postulated, treatment strategies to overcome resistance have not been established. We used a new synthetic retinoid, Am-80, as reinduction therapy for APL relapse after from ATRA-induced CR. Am-80 was several times more potent than ATRA in inducing differentiation in vitro. At a 6 mg/m2 dose, there were 24 evaluable patients; 14 (58%) achieved CR between days 20 and 58 (median, 37 days). Clinical response correlated with the in vitro response to Am-80. Adverse effects included retinoic acid syndrome (n = 1), hyperleukocytosis (n = 1), xerosis (n = 9), cheilitis (n = 8), hypertriglyceridemia (n = 16), and hypercholesterolemia (n = 15). Am-80 is active in APL after relapse from ATRA-induced CR. Further clinical trials are needed to establish strategies to overcome ATRA resistance.     

Effect of acute acipimox administration on the rates of lipid and glycogen synthesis in cachectic tumor-bearing rats

2'-Deoxycoformycin (dCF), a specific and potent inhibitor of adenosine deaminase, has demonstrated significant antitumor effect on lymphoid malignancies. The drug induced functional and morphologic differentiation of myeloid leukemia cells in combination with 2'-deoxyadenosine (dAd), but not dCF alone. NB4, a cell line derived from a patient with t(15; 17) acute promyelocytic leukemia (APL) underwent granulocytic differentiation when treated with all-trans retinoic acid (ATRA) or dCF plus dAd, but not with cytosine arabinoside. Pre-exposure of NB4 cells to ATRA greatly potentiated differentiation induced by dCF plus dAd, but pretreatment with dCF plus dAd before exposure to ATRA was less effective. Differentiation of NB4 cells was effectively induced by clinically applicable concentrations of dCF in combination with dAd. These findings may provide useful information about induction of differentiation in vivo.     

Modulation of homeobox B6 and B9 genes expression in human leukemia cell lines during myelomonocytic differentiation

Homeobox genes (HOX) may have a regulatory function in the differentiation process of hematopoiesis. We examined the change of HOX B6 and HOX B9 mRNA expressions during the in vitro differentiation of four myeloid leukemia cell lines because HOX B6 may be involved closely in myeloid differentiation. HL-60, NB4, NKM-1 and NOMO-1 were established from acute leukemia of M2, M3, M2 and M5 subtype of the French-American-British classification, respectively. All-trans retinoic acid (ATRA), TPA, and G-CSF were used as differentiation inducers. Each cell line was cultured with each inducer and total RNA was isolated on day 1, 2, 3, or 5. HOX B mRNA was detected by Northern blotting and RT-PCR methods. HOX B6 and HOX B9 mRNAs were constitutively expressed in NB4, NKM-1 and NOMO-1, but were expressed at very low levels in HL-60. HOX B6 and HOX B9 mRNAs were also expressed in fresh acute myelocytic leukemia blasts. HOX B6 mRNA expression in HL-60, NB4, and NKM-1 cultured with ATRA increased on day 3 and decreased on day 5. HOX B6 mRNA expression in NB4 and NKM-1 cultured with TPA decreased on day 3. HOX B9 mRNA expression displayed changes similar to those of HOX B6 mRNA in NB4 and NKM-1. These results indicate that myeloid leukemia cell lines express HOX B6 and HOX B9, and that their respective mRNA expressions in NB4 and HL-60 increase at a mid stage of myeloid differentiation by ATRA induction and then decrease during a late stage. HOX B6 mRNA expression decreased in monocytoid differentiation by TPA induction in NB4, HL-60 and NKM-1. HOX B6 antisense-oligonucleotide inhibited the proliferation of NB4 and NKM-1. These results suggest that HOX B gene expression is related to simultaneous activation of cellular proliferation and differentiation in leukemic cells.     

Differentiation of t(5;17) variant acute promyelocytic leukemic blasts by all-trans retinoic acid

All-trans retinoic acid (ATRA) induces differentiation of acute promyelocytic leukemic (APL) blasts from patients with t(15;17) APL. However, blasts from patients with the t(11;17) variant do not differentiate in response to ATRA. Our group has identified a variant of APL characterized by t(5;17) and expression of the NPM-RAR fusion gene product. From case reports it has been difficult to establish whether ATRA induces clinical responses in patients with this variant. In order to determine whether t(5;17) blasts differentiate with ATRA, we harvested mononuclear bone marrow cells from a patient with t(5;17) APL at time of relapse and cultured them in medium containing ATRA. Morphologic analysis of cytospins after 7 days of culture revealed that 60% of cells in the ATRA-treated culture had differentiated into mature neutrophilic forms, as opposed to less than 1% in the control culture. Seventy-three percent of cells acquired NBT positivity after exposure to ATRA, compared with 1% in the control culture. These results indicate that t(5;17) blasts retain the ability to terminally differentiate in response to retinoic acid.     

Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells

We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication.     

Neplanocin A, a potent inhibitor of S-adenosylhomocysteine hydrolase, potentiates granulocytic differentiation of acute promyelocytic leukemia cells induced by all-trans retinoic acid

Several neplanocin A analogs were synthesized and their growth-inhibiting and differentiation-inducing activities on myelogenous leukemia cells were examined. An adenosine kinase-ineffective analog of neplanocin A was effective in inducing differentiation, suggesting that phosphorylation of the nucleoside is not essential for inducing the differentiation of leukemia cells. Neplanocin A induced functional and morphological differentiation of HL-60 cells, but did not effectively induce differentiation of NB4, a cell line derived from a leukemia patient with t(15;17). However, these cells have been known to undergo granulocytic differentiation upon treatment with all-trans retinoic acid (ATRA), and are used as a model for differentiation therapy in acute promyelocytic leukemia. Preexposure of NB4 cells to low concentrations of neplanocin A greatly enhanced the ATRA-induced differentiation of the cells, whereas representative antileukemic drugs such as cytosine arabinoside and daunomycin did not enhance this differentiation. A clinical strategy that combines intermittent treatment with neplanocin A analogs and a low dose of ATRA may increase the clinical response and decrease the adverse effects of ATRA.     

Potentiated maturation with a high proliferating activity of acute promyelocytic leukemia induced in vitro by granulocyte or granulocyte/macrophage colony-stimulating factors in combination with all-trans retinoic acid

All-trans retinoic acid (ATRA) induces differentiation of acute promyelocytic leukemia (APL), but the effect of cytokines regulating myeloid differentiation on ATRA-induced APL cells is poorly understood. In this study, maturation and proliferation of fresh APL cells were examined when induced in vitro by granulocyte or granulocyte/macrophage colony-stimulating factors (G-CSF or GM-CSF) in combination with ATRA. APL cells showed a low proliferating activity when induced by ATRA alone. In contrast, cells induced by G-CSF or GM-CSF alone showed increased DNA syntheses, the levels of which were not significantly affected by the combination of ATRA with CSFs. Interestingly, G-CSF or GM-CSF potentiated the capability of ATRA-induced cells to reduce nitroblue tetrazolium (NBT), while G-CSF or GM-CSF alone induced no NBT reduction. Furthermore, in several patients examined, APL cells induced by ATRA with G-CSF showed an increased activity of chemotaxis and CD11a expression. These findings suggest that G-CSF or GM-CSF can potentiate differentiation of ATRA-induced APL cells while stimulating their proliferating activity as well, and that G-CSF, rather than GM-CSF, may be a useful adjunct to promote ATRA-induced differentiation of APL.     

The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.     

Inhibition of IFN-stimulated gene expression and IFN induction of cytolytic resistance to natural killer cell lysis correlate with E1A-p300 binding

Treatment of target cells with IFN induces resistance to NK cell lysis. This process is blocked by expression of E1A gene products in adenovirus (Ad)-infected and Ad-transformed cells. We compared the ability of adenovirus serotype 5 (Ad5) E1A exon 1 mutants to inhibit the induction of cytolytic resistance by IFN and block IFN-stimulated gene expression with their capacity to bind the cellular proteins p105 (retinoblastoma gene product), p107, and p300. E1A mutants that did not express conserved region 3 (CR3; residues 138-184) or contained deletions in the nonconserved regions between residues 26-35 or 86-120, bound p105, p107, and p300 and were not impaired in their capacity to block IFN-stimulated gene expression or IFN's induction of cytolytic resistance. E1A mutants with deletions in CR2 (residues 121-138) could not bind p105 or p107, but blocked IFN-stimulated gene expression and IFN's induction of cytolytic resistance. In contrast, mutants in CR1 or the N-terminal nonconserved region (residues 2, 4-25, and 48-60), which define E1A's binding site for p300, were unable to block either IFN-stimulated gene expression or IFN's induction of cytolytic resistance. We conclude that E1A's capacity to block both IFN-stimulated gene expression and IFN's induction of cytolytic resistance appears to be transduced through a pathway that involves E1A-p300 binding. The capacity of E1A to block IFN's induction of cytolytic resistance is probably secondary to E1A's more general ability to inhibit IFN-stimulated gene expression.     

4-Oxoretinol, a metabolite of retinol in the human promyelocytic leukemia cell line NB4, induces cell growth arrest and granulocytic differentiation

All-trans-retinoic acid (RA) is used as a differentiation therapy for acute promyelocytic leukemia. Patients can become resistant to RA, and this resistance is thought to be mediated in part by an increase in the rate of RA metabolism. We have characterized the metabolism of all-trans-retinol (ROL; vitamin A) in NB4 cells, which are human promyelocytic leukemia cells. NB4 cells metabolize ROL into a variety of compounds, including all-trans-4-hydroxyretinol, all-trans-4-oxoretinol (4-oxoROL), 14-hydroxy-4,14-retro-retinol, anhydroretinol, and several ROL esters. No metabolism of ROL to RA or to RA derivatives in NB4 cells was detected. The rate of ROL metabolism increased after cell differentiation; in a 24-h period, differentiated cells metabolized 2-fold more ROL than did undifferentiated cells. The major difference in the ROL metabolism pattern between undifferentiated and differentiated cells was an approximately 10-fold increase in the production of all-trans-4-hydroxyretinol and 4-oxoROL in differentiated cells. Furthermore, exogenously added 4-oxoROL was capable of eliciting NB4 cell differentiation, as measured by growth inhibition, nitroblue tetrazolium reduction, nuclear body relocalization of PML, and surface expression of CD11b. In addition, 4-oxoROL synergized with IFN-gamma in the promotion of NB4 cell growth arrest. Following treatment of NB4 cells with 4-oxoROL to induce differentiation, the production of 4-oxoROL from ROL was observed; this indicated that 4-oxoROL induces its own synthesis in NB4 cells. In addition, 48 h after the administration of 1 microM 4-oxoROL, NB4 cells maintained a high intracellular concentration (17 microM) of 4-oxoROL. These unique properties of 4-oxoROL may provide advantages over RA in the treatment of promyelocytic leukemia cells because it may be possible to maintain cytodifferentiating concentrations of 4-oxoROL in the cells for extended periods of time.     

19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.     

Wt-p53 action in human leukaemia cell lines corresponding to different stages of differentiation

Recent studies support the potential application of the wt-p53 gene in cancer therapy. Expression of exogenous wt-p53 suppresses a variety of leukaemia phenotypes by acting on cell survival, proliferation and/or differentiation. As for tumour gene therapy, the final fate of the neoplastic cells is one of the most relevant points. We examined the effects of exogenous wt-p53 gene expression in several leukaemia cell lines to identify p53-responsive leukaemia. The temperature-sensitive p53Val135 mutant or the human wt-p53 cDNA was transduced in leukaemia cell lines representative of different acute leukaemia FAB subtypes, including M1 (KG1), M2 (HL-60), M3 (NB4), M5 (U937) and M6 (HEL 92.1.7), as well as blast crisis of chronic myelogenous leukaemia (BC-CML: K562, BV173) showing diverse differentiation features. By morphological, molecular and biochemical analyses, we have shown that exogenous wt-p53 gene expression induces apoptosis only in cells corresponding to M1, M2 and M3 of the FAB classification and in BC-CML showing morphological and cytochemical features of undifferentiated blast cells. In contrast, it promotes differentiation in the others. Interestingly, cell responsiveness was independent of the vector used and the status of the endogenous p53 gene.